PEGylated Recombinant Human Growth Hormone Compounds

ABSTRACT

A chemically modified human Growth Hormone (rhGH) prepared by attaching a transient linker which comprises a polyethylene glycol. The chemically modified protein may have a much longer lasting rhGH activity than that of the unmodified rhGH, enabling reduced dose and scheduling opportunities and the modified rhGH may not cause lipoatrophy. Also includes methods of use for the treatment and/or prevention of diseases or disorders in which use of growth hormone is beneficial.

The present application is a continuation application of U.S. patentapplication Ser. No. 12/990,101 filed on Dec. 22, 2010, the disclosureof which is incorporated herein by reference in its entirety, whichclaims priority from PCT Patent Application No. PCT/EP2009/0565194 filedon Apr. 29, 2009, which claims priority from European PriorityApplication No. 08167289.1 filed on Oct. 22, 2008, European PriorityApplication No. 08162865.3 filed on Aug. 22, 2008, and European PriorityApplication No. 08155408.1 filed on Apr. 29, 2008.

FIELD OF THE INVENTION

This invention relates to a pharmaceutical composition comprisingsuitable pharmaceutical excipients and also comprising a clinicallyeffective amount of a recombinant human growth hormone (rhGH) PEGylatedprodrug which can be administered less frequently than available humangrowth hormone products and may not cause injection side lipoatrophy.The present invention also relates to such prodrugs.

BACKGROUND ART

Growth hormone (GH) is a hormone that stimulates growth and cellreproduction in humans and other animals. It is a 191-amino acid, singlechain polypeptide hormone which is synthesized, stored, and secreted bythe somatotroph cells within the lateral wings of the anterior pituitarygland. The hormone is also known as somatotropin when referring togrowth hormone produced by recombinant DNA technology, and isabbreviated “rhGH”.

Growth hormone has a variety of functions in the body, the mostnoticeable of which is the increase of height throughout childhood, andthere are several diseases which can be treated through the therapeuticuse of GH.

The effects of growth hormone deficiency vary depending on the age atwhich they occur. In children, growth failure and short stature are themajor manifestations of GH deficiency. It can also cause sexualimmaturity. In adults the effects of deficiency are more subtle, and mayinclude deficiencies of strength, energy, and bone mass, as well asincreased cardiovascular risk.

There are many causes of GH deficiency, including mutations of specificgenes, congenital malformations involving the hypothalamus and/orpituitary gland, and damage to the pituitary from injury, surgery ordisease.

Deficiency is treated through supplementation with external GH. All GHin current use is a biosynthetic version of human GH, manufactured byrecombinant DNA technology. GH is used as replacement therapy inchildren and adults with GH deficiency of either childhood-onset (aftercompleting growth phase) or adult-onset (usually as a result of anacquired pituitary tumor). In these patients, benefits have variablyincluded reduced fat mass, increased lean mass, increased bone density,improved lipid profile, reduced cardiovascular risk factors, andimproved psychosocial well-being.

Genentech Inc (US) was the first to clone rhGH and this was described inpatent EP-B 22242. As of 2006, synthetic growth hormones available inthe United States and Europe (and their manufacturers) included Nutropin(Genentech), Humatrope (Eli Lilly), Genotropin (Pfizer), Norditropin(Novo Nordisk), Saizen (Merck Serono), and Omnitrope (Sandoz).

Although molecular biological techniques have dramatically increased theavailability of many proteins and/or polypeptides (hereinafter referredto as proteins), the therapeutic use of said proteins is often timeshindered by other factors, such as short plasma half-life due to renaland receptor-mediated clearance, aggregation, proteolytic degradation,poor bioavailability and physical properties which preclude efficientformulations.

A mechanism for enhancing protein availability is by conjugation of theprotein with derivatizing compounds, which include, but are not limitedto, polyethylene glycol and polypropylene glycol. Some of these benefitsrecognized include: lowered immunogenicity and antigenicity, increasedduration of action, and altered pharmacokinetic properties. [Veronese,F. M. “Enzymes for Human Therapy: Surface Structure Modifications,”Chimica Oggi, 7:53-56 (1989)] (Herein reference 5).

By PEGylating rhGH, it may be possible to improve the characteristics ofthe molecule for medical use by increasing its in vivo half-life (herebyachieving reduced dosage or reduced frequency of dosing), improving itsstability and decreasing its antigenicity or a combination thereof.

Generally, this type of modification to a molecule is well known in theart and there are numerous patents available in the patent literature,describing this concept. For example a PEGylated Erythropoietin (EPO)from Hofmann La Roche is described in EP-B 1196443 claiming a specificlinker comprising PEG covalently bound to EPO, a PEGylated interferonalpha described in EP-B 975369 from the company Nektar/La Roche andanother PEGylated interferon alpha in EP-B 1562634 from the companyHofmann La Roche.

In vivo clearance of rhGH is believed to occur by the following twomechanisms. The first is renal clearance where rhGH is cleared from thecirculation by renal glomerular filtration. Renal clearance of rhGH iswell documented and PEGylation of synthetic rhGH is therefore an obviouschoice to solve this problem. Renal clearance accounts for around 25-53%of the total clearance of rhGH (Girard, J. Mehls, O. J. Clin. Invest.1994 March; 93(3): 1163-1171, reference 3 herein.)

The second mechanism is hepatic clearance (liver). Hepatic GH uptakeoccurs by receptor-mediated endocytosis followed by lysosomaldegradation.

A third mechanism is receptor mediated clearance in other tissue such aschondrocytes of the cartilage. By reducing the binding affinity of GH tothe GH receptor by PEGylation, the receptor mediated clearance will bereduced.

However, there are dedicated problems with the administration of rhGH.One major disadvantage of subcutaneously administrated rhGH is theoccurrence of lipoatrophy in patients receiving the treatment.

Lipoatrophy is the medical term used for localized loss of fat tissue.Subcutaneously administered rhGH formulations have displayed lipoatrophyproblems, which is believed to be caused by high local concentration ofthe growth hormone complex and at the injection site.

Büyükgebiz A. et al published in J. Pediatr. Endocrinol. Metab. 1999January-February; 12(1):95-7 describes such a medical record (hereinreference 1). This is a report of a patient with isolated GH deficiencydue to 6.7 kb gene deletion who received high dose rhGH treatment anddeveloped local lipoatrophies at injection sites without any antibodydetection after 6 years of therapy. The etiology of the lipoatrophy issuspected to be by the direct lipolytic effect of high doses of rhGH.

Lipoatrophy related to the administration of rhGH is believed to becaused by the rhGH activity itself, by higher concentrations and byprolonged exposure. These higher concentrations occur near injectionssites.

The chance that high growth hormone activity accumulates near theinjection site is even higher in case that rhGH is PEGylated because ofan increased residence time. In the case of PEGylated rhGH formulations,the tissue will experience a sustained and increased exposure to growthhormone activity, due to the fact that the PEGylated conjugate possessactivity necessary for pharmacological activity and the conjugate isdiffusion limited due to the conjugate size. The outcome is increasedlipolysis at the injection site.

WO-A 2005/079838 describes pegylated hGH, wherein the hGH moiety isattached to a polyethylene glycol polymer via amino functional group,which can be considered as permanent attachment due to the stability ofthe amino group. An example of such a PEGylated hGH compound, whichexhibits lipoatrophy, is the compound PHA-794428. Compound PHA-794428 isa PEGylated rhGH and also described in WO-A 2 5 2005/079838 from thecompany Pharmacia (acquired by Pfizer) and further described in Horm.Res. 2006; 65 (suppl. 4): 1-213, CF1-98 GH/IGF Treatment with title“First in-human study of PEGylated recombinant human growth hormone”,Philip Harris et al. (herein reference 4).

According to the clinical trial information as published onwww.clinicaltrials.gov, the trial was terminated on 10 Dec. 2007.Pfizer's decision to terminate the program was due to cases ofinjection-site lipoatrophy that were reported in the clinical Phase 2studies after a single injection of PHA 794428.

WO-A 2006/102659 (Nektar) also describes and suggests rhGH-PEGconjugates (linear and branched types) via amide bond. The problem to besolved in WO-A 2006/102659 is described in paragraph [0005] on page 2.According to the applicant, the problem to be solved is reduced dosingfrequency. Since rhGH therapy typically requires daily injections,patients, and in particular, pediatric patients, dislike theinconvenience and discomfort associated with this regimen. The solutiondescribed in Nektar's WO-A is the provision of new PEG-rhGH conjugates.

In table 6, [0257] of the WO-A it can be seen that the PEG-rhGHconjugates have a relatively low activity in vitro as compared to thenative growth hormone without PEG. Despite the low in vitro activities,the PEGylated rhGH conjugates were active in vivo. In relation to thisreads section [0261]: “Although the preliminary in vitro results suggestthat increasing the amount of PEG attached to hGH reduces its ability tostimulate the hGH receptor, based on the preliminary in vivo results, itappears that a reduction in bioactivity is more than balanced byincreased half-life and/or plasma availability, thus leading to aconclusion that the conjugates provided herein possess a superiorpharmacodynamic effect in vivo when compared to unmodified rhGH at anidentical dosing regimen”.

WO-A 2006/102659 (Nektar) does not describe auto-cleavable linkers—i.e.it is simply observed that PEG-rhGH conjugates are active in vivoalthough their in vitro activities are significantly reduced. Theproblem of lipoatrophy is not addressed.

A solution to the challenge of engineering the desired properties ofreduced lipoatrophy and reduced injection frequency into a PEGylatedconjugate of hGH is the use of a prodrug approach. A prodrug is anycompound that undergoes biotransformation before exhibiting itspharmacological effects. Prodrugs can thus be viewed as drugs containingspecialized non-toxic protective groups used in a transient manner toalter or to eliminate undesirable properties in the parent molecule. Inthis case, a polymeric carrier would transiently reduce the activity ofgrowth hormone and consequently reduce the likelihood of tissuelipolysis. Transient conjugation to a polymeric carrier would at thesame time extend the half-life of the conjugate and therefore providefor a long-acting delivery of hGH.

Numerous macromolecular prodrugs are described in the literature wherethe macromolecular carrier is linked via a labile ester group to themedicinal agent (e.g. Y. Luo, M R Ziebell, G D Prestwich, “A HyaluronicAcid—Taxol Antitumor Bioconjugate Targeted to Cancer Cells”,Biomacromolecules 2000, 1, 208-218, J Cheng et al, Synthesis of Linear,beta-Cyclodextrin Based Polymers and Their Camptothecin Conjugates,Bioconjugate Chem. 2003, 14, 1007-1017, R. Bhatt et al, Synthesis and inVivo Antitumor Activity of Poly(L-glutamic acid) Conjugates of20(S)-Camptothecin, J. Med. Chem. 2003, 46, 190-193; R. B. Greenwald, A.Pendri, C. D. Conover, H. Zhao, Y. H. Choe, A. Martinez, K. Shum, S.Guan, J. Med. Chem., 1999, 42, 3657-3667; B. Testa, J. M: Mayer inHydrolysis in Drug and Prodrug Metabolism, Wiley-VCH, 2003, Chapter 8)In theses cases, the conjugated functional group of the bioactive entityis a hydroxyl group or a carboxylic acid.

Especially for biomacromolecules but also for small molecule polymerprodrugs, it may be desirable to link the macromolecular carrier toamino groups (i.e. N-terminus or lysine amino groups of proteins) of thebioactive entity. This will be the case if masking the drug'sbioactivity requires conjugation of a certain amino group of thebioactive entity, for instance an amino group located in an activecenter or a region or epitope involved in receptor binding. Also, duringpreparation of the prodrug, amino groups may be more chemoselectivelyaddressed and serve as a better handle for conjugating carrier and drugbecause of their greater nucleophilicity as compared to hydroxylic orphenolic groups. This is particularly true for proteins which maycontain a great variety of different reactive functionalities. In thiscase non-selective conjugation reactions lead to undesired productmixtures which require extensive characterization or purification andmay decrease reaction yield and therapeutic efficiency of the product.

Prodrug activation may occur by enzymatic or non-enzymatic cleavage ofthe labile bridge between the carrier and the drug molecule, or asequential combination of both, i.e. an enzymatic step followed by anonenzymatic rearrangement.

In WO-A 2005/099768 PEGylated linker molecules with auto-cleavablelinkers for a large group of biomolecules including somatropins (claim6) are described. In WO-A 2005/099768, the problem to be solved is theinterpatient variability and unpredictable effect of prodrug activationwhen enzymatic mechanism is involved (page 12, line 17-30). Thisapplication describes as a solution an aromatic linker, which may be PEGbased. This linker-PEG binds the drug in a way that the drug activity issignificantly reduced. It is activated only on release of the drug,which is initiated by hydrolysis. The hydrolysis rate can be controlledchemically. No special emphasis is given on GH and relevant problems,like lipoatrophy, in relation to this as such.

In summary, none of the above mentioned citations describes a solutionto develop a long-acting rhGH, based on a prodrug conjugate that can beadministered less frequently without increasing the frequency oflipoatrophy.

Thus an object of the present invention is the provision of such aprodrug or a pharmaceutical composition comprising said prodrug toreduce the administration frequency of rhGH using PEG conjugated to rhGHwithout significantly inducing lipoatrophy.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a pharmaceutical compositioncomprising suitable pharmaceutical excipients and also comprising ahuman in vivo clinical effective amount of a recombinant human growthhormone rhGH PEGylated prodrug conjugate, wherein PEG is linked to rhGHvia a self hydrolysable (autocleavage) transient linker; said prodrugconjugate is characterized in that:

-   -   (1): the conjugate has a GH activity which is less than 5% of        the native growth hormone without PEG; and    -   (2): the linker autohydrolysis rate is such that the in vivo        half-life is from 10 hours to 600 hours.

Property (1) ensures that the prodrug has a low incidence of lipoatrophydespite having a significantly extended duration of action in vivo.Without being limited by theory the present inventors believe that ifthe prodrug had a higher GH activity, this product would still inducelipoatrophy at a higher frequency than currently marketed rhGH products.

Property (2) ensures that rhGH (without PEG) is released gradually overtime so one can administrate the rhGH pharmaceutical product lessfrequently than human growth hormone, e.g. only once weekly or oncemonthly instead of daily administrations, while still retaining fullefficacy compared to rhGH.

Preferably, the in vivo half life is up to 5 times shorter, e.g. 2, 3,4, or 5 times shorter, than the corresponding hGH PEGylated prodrugconjugate's in vitro half-life. More preferably, the in vivo half-lifeis up to 3 times shorter than the corresponding hGH PEGylated prodrugconjugate's in vitro half-life. Most preferably, the in vivo half-lifeis up to 2 times shorter than or almost identical to the correspondinghGH PEGylated prodrug conjugate's in vitro half-life.

This invention applies to rhGH PEGylated prodrugs, in particular to rhGHPEGylated carrier prodrugs including cascade carrier prodrugs.

Prodrugs may be defined as therapeutic agents that are inactive per sebut are predictably transformed into active metabolites (see B. Testa,J. M: Mayer in Hydrolysis in Drug and Prodrug Metabolism, Wiley-VCH,2003, page 4). In carrier prodrug systems, many medicinal agents areinactive or show decreased biological activity when a polymer iscovalently conjugated to the drug molecule. In these cases, a transientlinkage of drug and carrier is applied in such a fashion that themedicinal agent is released from the polymeric carrier in vivo to regainits biological activity. The reduced biological activity of the prodrugas compared to the released drug is of advantage if a slow or controlledrelease of the drug is desired. In this case, a relatively large amountof prodrug may be administered without concomitant side effects and therisk of overdosing. Release of the drug occurs over time, therebyreducing the necessity of repeated and frequent administration of thedrug.

In polymeric carrier prodrugs, the biologically active moieties areoften linked to a polymeric carrier moiety by a labile bridge formedbetween the carrier moiety and a functional group of the drug molecule.Cleavage of a carrier prodrug generates a molecular entity (drug) ofincreased bioactivity and at least one side product, the carrier. Thisside product may be biologically inert (for instance PEG). Aftercleavage, the bioactive entity will reveal at least one previouslyconjugated and thereby masked or protected functional group, and thepresence of this group typically contributes to the bioactivity.

The GH activity can be measured using methods known in the art. In thisrespect emphasis is made to example 1. Based on the fact that sometransient linkers applicable for the present invention may have an invitro half-life of less than 3000 h the respective GH activitymeasurement is made indirectly by determining the GH activity of arespective PEG conjugate comprising a permanent linker instead of thetransient linker. This can be carried out as WO2006102659 describes onpage 74 paragraph 0240, the biological activity of rhGH and theconjugates described herein shall be assessed in vitro using an NB2-IIrat lymphoma cell proliferation assay. Briefly, NB2-II cells derivedfrom a rat lymphoma are incubated with rhGH, which lead to binding ofthe rhGH molecule to its receptor on the cell surface. Receptor bindinginduces the signal transduction cascade, which results in proliferationof the cells. Assay results are based on determined protein content, anda 100% bioactivity of unmodified rhGH.

Preferably, for the measurement of the GH activity the protocol asdescribed in Example 24 is used.

The in vitro half life can be measured using methods known in the art.In this respect emphasis is made to example 2.

Accordingly, a second aspect of the invention relates to a clinicaleffective amount of the pharmaceutical composition comprising the rhGHPEGylated prodrug of the first aspect for use in a method for treatmentof a GH related disease in a human person.

This second aspect may alternatively be formulated as a method fortreatment of a GH related disease in a human person comprisingadministrating to a human person a clinical effective amount of thepharmaceutical composition comprising the rhGH PEGylated prodrug of thefirst aspect.

In a situation, where the residual activity of the prodrug (as is thecase for transiently PEG conjugated rhGH) is significantly reduced ascompared to native human GH, lipolytic effects may not occur even atprolonged exposure.

The herein described compounds termed rhGH PEGylated prodrugs areconjugates that may have significantly reduced residual activitycompared to human GH. To exhibit therapeutically useful activity, rhGHhas to be released from the prodrug conjugate, for which the describedprodrugs herein need to undergo an activation step (e.g. 1,6-releasemechanisms), termed herein as autocleavage, cleaving the PEG group fromthe drug. The 1,6-release mechanism is well described in WO-A2005/099768.

Without being limited by theory, the present inventors believe that theherein disclosed transiently PEGylated rhGH conjugates significantlyreduce lipoatrophy because of the low activity of the PEGylated rhGHconjugates, before PEG is gradually cleaved off by the autocleavablelinker. This may ensure that the prodrugs will not induce lipoatrophymore frequently than human GH or other permanently PEGylated rhGHcompounds as described above.

The problem underlying the present invention is also solved by apharmaceutical composition comprising suitable pharmaceutical excipientsand also comprising a prodrug conjugate of the human growth hormone(hGH) of formula (AA)

hGH-NH-L^(a)-S⁰   (AA),

wherein

hGH-NH represents the hGH residue;

L^(a) represents a functional group, which is self hydrolysable(auto-cleavable) by an auto-cleavage inducing group G^(a); and

S⁰ is a polymer chain having a molecular weight of at least 5 kDa andcomprising an at least first branching structure BS¹, the at least firstbranching structure BS¹ comprising an at least second polymer chain S¹having a molecular weight of at least 4 kDa, wherein at least one of S⁰,BS¹, S¹ further comprises the auto-cleavage inducing group G^(a) andwherein the branching structure BS¹ further comprises an at least thirdpolymer chain S² having a molecular weight of at least 4 kDa or at leastone of S⁰, S¹ comprises an at least second branching structure BS²comprising the at least third polymer chain S² having a molecular weightof at least 4 kDa and wherein the molecular weight of the prodrugconjugate without the hGH-NH is at least 25 kDa and at most 1000 kDa,preferably at least 25 kDa and at most 500 kDa, even more preferably atleast 30 kDa and at most 250 kDa, even more preferably at least 30 kDaand at most 120 kDa, even more preferably at least 40 kDa and at most100 kDa, even more preferably at least 40 kDa and at most 90 kDa.

Surprisingly it was found, that the residual activity of a prodrug ofthe present invention can be efficiently reduced by providing apolymeric carrier having at least 3 chains of a certain minimum length(as defined by their molecular weight) and thus, in combination with atransient linker as described herein solve the problem of providing anhGH prodrug that can be administered less frequently without increasingthe risk of lipoatrophy. The prodrug should therefore be water-soluble.

For at least bis-conjugated prodrugs the problem can be solved by apharmaceutical composition comprising suitable pharmaceutical excipientsand also comprising a prodrug conjugate of the human growth hormone(hGH) of formula (AB))

hGH-(NH-L-S⁰)_(n)   (AB),

wherein

n is 2, 3, or 4; preferably 2;

hGH(-NH)_(n) represents the hGH residue;

each L is independently a permanent functional group L^(p); or afunctional group L^(a), which is self hydrolysable (auto-cleavable) byan auto-cleavage inducing group G^(a); and

each S⁰ is independently a polymer chain having a molecular weight of atleast 5 kDa, wherein S⁰ is optionally branched by comprising an at leastfirst branching structure BS¹, the at least first branching structureBS¹ comprising an at least second polymer chain S¹ having a molecularweight of at least 4 kDa, wherein at least one of S⁰, BS¹, S¹ furthercomprises the auto-cleavage inducing group G^(a) and wherein themolecular weight of the prodrug conjugate without the hGH-NH is at least25 kDa and at most 1000 kDa, preferably at least 25 kDa and at most 500kDa, even more preferably at least 30 kDa and at most 250 kDa, even morepreferably at least 30 kDa and at most 120 kDa, even more preferably atleast 40 kDa and at most 100 kDa, even more preferably at least 40 kDaand at most 90 kDa.

Yet another aspect of the present invention is a prodrug conjugate asdefined above.

Preferred embodiments of the present invention are described below, byway of examples only.

Definitions

Prior to a discussion of the detailed embodiments of the invention isprovided a definition of specific terms related to the main aspects ofthe invention.

In general, all specific technical terms used herein shall be understoodas the skilled person would understand them in the present technicalcontext.

rhGH or hGH or GH or hGH residue refers to human growth hormone. NH-hGHis a hGH residue, wherein the —NH— of —NH-hGH represents an amino groupof hGH.

The term “activity” herein is understood as the ability of growthhormone or a conjugate thereof, to evoke a biological response whenadministered to a mammal, e.g. in an in vivo model, or to produce ameasurable response in an in vitro model as described in the examples.

In a prodrug system, measured activity will have two contributions, onefrom the released free drug entity and one from the not yet cleavedprodrug conjugate. In order to differentiate the activity of the prodrugconjugate, the term “residual activity” herein is understood as theportion of the measured prodrug activity that may be attributed to theprodrug conjugate.

The term “autocleavage” herein is understood as rate-limiting hydrolyticcleavage of the bond between the transient linker and the drug moleculerhGH in an aqueous buffered solution under physiological conditions ofpH 7.4 and 37° C. Autocleavage does not require the presence of enzyme.This auto-cleavage or self hydrolysis is controlled by an auto-cleavageinducing group, which is part of the prodrug molecule. Thisauto-cleavage inducing group may be present as such or in a masked formso that unmasking is required before the self hydrolysis mechanism canstart.

Linker autohydrolysis rate refers to the rate of cleavage of ahGH-PEGylated prodrug in vivo. As enzymatic or other effects almostalways cause prodrug linker hydrolysis to proceed faster in vivo than invitro, it is defined that a hGH PEG prodrug cleaves in an autohydrolyticfashion if the prodrug's in vivo half-life is up to 5 times shorter thanthe corresponding hGH PEGylated prodrug conjugate's in vitro half-life.

The term “transient linkage” or “transient linker” herein is understoodas describing the lability of the linkage between PEG and rhGH in a rhGHPEGylated prodrug. In such transient linkages, rhGH is released from thecorresponding prodrug with an in vivo linker half-life of up to 1200hours.

The term “conjugate” herein is understood as one or more PEG moleculescovalently bound to the drug herein being human growth hormone.

The term “transient conjugate” refers to hGH PEGylated prodrugscontaining at least one transient linkage.

The term “permanent conjugate” refers to hGH PEGylated conjugates orprodrugs where the PEG polymer is connected to hGH by means of linkageswith an in vitro half-life of at least 3000 hours.

In vitro half-life or in vitro linker half-life is the release of 50% ofhGH from hGH PEGylated prodrug in buffer at pH 7.4 and 37° C.

The terms “in vivo half life” or “in vivo linker half-life” areunderstood as the time interval in which 50% of the initial proportionof the growth hormone is released from the hGH PEGylated prodrug afteradministration to the human body, calculated by taking into account thecompound's corresponding conjugate half-life as described in example 2.

The term “conjugate half life” is understood as the time interval inwhich 50% of a hGH PEGylated permanent conjugate as defined above iscleared from the blood circulation.

The term “lipoatrophy” herein is understood as a medical term forlocalized loss of fat tissue. In the present context “lipoatrophy”refers to injection site lipoatrophy meaning tissue lipolysis occurringin close proximity of the injection site.

The term “prodrug” herein is understood is any compound that undergoestransformation before exhibiting its full pharmacological effects.Classification of prodrug systems is given by under IUPAC definitions(http://www.chem.qmul.ac.uk/iupac/medchem, accessed on 8 Mar. 2004):

Prodrug

A prodrug is any compound that undergoes biotransformation beforeexhibiting its pharmacological effects. Prodrugs can thus be viewed asdrugs containing specialized non-toxic protective groups used in atransient manner to alter or to eliminate undesirable properties in theparent molecule.

Double Prodrug (or Pro-Prodrug)

A double prodrug is a biologically inactive molecule which istransformed in vivo in two steps (enzymatically and/or chemically) tothe active species.

Carrier-Linked Prodrug (Carrier Prodrug)

A carrier-linked prodrug is a prodrug that contains a temporary linkageof a given active substance with a transient carrier group that producesimproved physicochemical or pharmacokinetic properties and that can beeasily removed in vivo, usually by a hydrolytic cleavage.

Cascade Prodrug

A cascade prodrug is a prodrug for which the cleavage of the carriergroup becomes effective only after unmasking an activating group.

Biotransformation

Biotransformation is the chemical conversion of substances by livingorganisms or enzyme preparations.

Correspondingly, a cascade autohydrolysis-inducing group becomeseffective only after unmasking of certain autohydrolysis-inducingstructural elements. There may be one or more cascade unmasking stepsrequired to reveal the autohydrolyis-inducing structural elements. Atleast one of the unmasking steps may be based on a biotransformationstep.

The term “a pharmaceutical composition comprising a human in vivoclinical effective amount of a recombinant human growth hormone (rhGH)PEGylated prodrug” is to be understood as an amount that is sufficientlyhigh to obtain a wanted clinical effect in a human after administrationof the pharmaceutical composition to the human—e.g. a wanted clinicaleffect in relation to treatment of a GH related disease. In the presentcontext the skilled person routinely is able to adjust the amount ofrecombinant human growth hormone (rhGH) PEGylated prodrug to beadministrated in order to get a wanted clinical effect.

The term “physiological condition” herein is understood as any in vitroor in vivo condition, identical or resembling, the pH and temperatureconditions in the human body. More specifically physiological conditionsis referring to conditions at around pH 7.4 (pH 6.8 to pH 7.8) and about37° C. (35° C. to 40° C.).

The term “linker” is frequently used in publications in the field ofbioconjugation and broadly describes chemical structures used to connecttwo molecular entities. Such connectivity may be of permanent ortransient nature.

A transient linker is a linker in which the conjugation of drug to PEGmolecule is reversible. This implies that cleavage of the linkerreleases the drug in its native and active form. To structurallydifferentiate a transient linker unit from the polymer carrier may bedifficult in the case of carrier prodrugs, particularly if the polymeris permanently attached to the linker and the linker-related degradationproduct is therefore not released as a consequence of prodrug cleavage.Structural characterization of a linker is even more challenging if thelinker is functioning both as an auto-cleavage inducing group and abranching unit. Therefore, within the meaning of the present invention,the term linker may be used synonymous with a combination of afunctional group La and an auto-cleavage inducing group G^(a). In caseswhere carrier prodrugs are described where the carrier is a branchedPEG, it is preferred to use structural descriptions based oncombinations of L^(a) and G^(a). In such case, the cleavage-inducinggroup G^(a) is considered to be part of the carrier polymer. Variationof the chemical nature of G^(a) allows the engineering of the propertiesof the self-cleaving properties of a corresponding carrier-linkedprodrug to a great extent.

The term “permanent linker” refers to a PEG conjugate to a hGH-donatedprimary amino group by formation of an aliphatic amide or aliphaticcarbamate. If conventional PEGylation reagents are used, resultingconjugates are usually very stable against hydrolysis and the rate ofcleavage of the amide or carbamate bond would be too slow fortherapeutic utility in a prodrug system. Nevertheless such permanentlinker conjugates are useful for the investigation of the therapeuticutility of a prodrug conjugate as they allow for assessment of residualactivity.

If such stable linkages are to be employed in a prodrug approach,cleavage of the functional group is not possible in a therapeuticallyuseful timeframe without enzymatic catalysis.

The term “water-soluble prodrug” means a prodrug that is soluble inwater under physiological conditions. Typically, a water-soluble prodrugwill transmit at least 75%, more preferably at least 95%, of lighttransmitted by the same solution after filtering. On a weight basis, awater soluble polymer will preferably be at least about 35% (by weight)soluble in water, still more preferably at least about 50% (by weight),still more preferably at least about 70% (by weight), still morepreferably at least about 85% (by weight), still more preferably atleast about 95% (by weight) or completely soluble in water.

The term “PEG” or “pegylation residue” is used herein exemplary forsuitable water-soluble polymers characterized by repeating units.Suitable polymers may be selected from the group consisting ofpolyalkyloxy polymers, hyaluronic acid and derivatives thereof,polyvinyl alcohols, polyoxazolines, polyanhydrides, poly(ortho esters),polycarbonates, polyurethanes, polyacrylic acids, polyacrylamides,polyacrylates, polymethacrylates, polyorganophosphazenes, polysiloxanes,polyvinylpyrrolidone, polycyanoacrylates, and polyesters.

PEG chains may consist of an interconnecting moiety, a polymer moiety,and an end group.

In case of branched monoconjugates of hGH PEGylated prodrugs, thecritical distance defines the shortest distance between the attachmentsite of PEG chain S⁰ to L^(a) and the first branching structure BS¹measured as connected atoms.

The term “PEG load” herein is understood as a descriptor of themolecular mass of a polymer chain consisting of a number of repeatingunits attached to hGH. Total PEG load is understood as the totalmolecular mass of all polymeric carrier chains attached to hGH on amolecular basis.

DETAILED DESCRIPTION OF THE INVENTION

Pharmaceutical Composition Comprising Suitable Pharmaceutical Excipients

As known to the skilled person a pharmaceutical composition comprisespharmaceutically acceptable excipients and/or carriers.

“Pharmaceutically acceptable” is meant to encompass any excipient and/oradditive, which does not interfere with the effectiveness of thebiological activity of the active ingredient and that, is not toxic tothe host to which it is administered.

In a preferred embodiment the pharmaceutical composition is acomposition for subcutaneous administration, intramuscularadministration or intravenous injection. This is examples of preferredadministration routes for treatment of a relevant disorder/disease asdescribed herein.

The pharmaceutical composition may comprise other active ingredientsthan an rhGH PEGylated prodrug as described herein.

Recombinant Human Growth Hormone (rhGH)

Since the recombinant human GH is identical in sequence to natural humanGH, the term recombinant human growth hormone (rhGH) relates herein alsoto so-called biogenerics equivalents. Thus, the terms rhGH and hGH canbe used synonymously within the meaning of the present invention.

The term “biogenerics” herein is understood generic forms ofbiopharmaceuticals; molecules developed using biological processes,usually through modern biotechnology activity. Generic chemicalpharmaceuticals can be defined as those molecules which, when comparedwith the originator product: have essentially similar activity, areessentially chemically identical to their branded counterparts, arebioequivalent, achieve market authorization through an abbreviatedprocedure following patent expiry.

As known to the skilled person, it is today routine work to make e.g.minor amino changes of a biologics of interest (herein GH) withoutsignificantly affecting the activity of the biologics.

Besides recombinant human and biogenerics, the term recombinant humangrowth hormone (rhGH) relates herein also to all possible rhGHpolypeptides.

A precise description of possible rhGH polypeptides is given in WO-A2005/079838 from the Pharmacia Corporation provided on page 15,paragraph 0043 till and including paragraph 0053.

The term “hGH polypeptide or hGH protein”, when used herein, encompassesall hGH polypeptides, preferably from mammalian species, more preferablyfrom human and murine species, as well as their variants, analogs,orthologs, homologs, and derivatives, and fragments thereof that arecharacterized by promoting growth in the growing phase and inmaintaining normal body composition, anabolism, and lipid metabolism.

The term “hGH polypeptide or protein” preferably refers to the 22 kDahGH polypeptide having a sequence as disclosed in A. L. Grigorian etal., Protein Science (2005), 14, 902-913 as well as its variants,homologs and derivatives exhibiting essentially the same biologicalactivity (promoting growth in the growing phase and in maintainingnormal body composition, anabolism, and lipid metabolism). Morepreferably, the term “hGH polypeptide or protein” refers to thepolypeptide having exactly the abovementioned sequence.

Derivatives of hGH encompass especially hGH prodrug conjugatescomprising permanently linked polymers, like PEG, i.e. the prodrug ofthe present invention may comprise in addition to one or more transientlinker polymer conjugates further permanent linker polymer conjugates.

The term “hGH polypeptide variants”, as used herein, refers topolypeptides from the same species but differing from a reference hGHpolypeptide. Generally, differences are limited so that the amino acidsequences of the reference and the variant are closely similar overalland, in many regions, identical. Preferably, hGH polypeptides are atleast 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to a referencehGH polypeptide, preferably the hGH polypeptide having a sequence asindicated in A. L. Grigorian et al., Protein Science (2005), 14,902-913. By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a query amino acid sequence, it is intendedthat the amino acid sequence of the subject polypeptide is identical tothe query sequence except that the subject polypeptide sequence mayinclude up to five amino acid alterations per each 100 amino acids ofthe query amino acid sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence. The query sequence may be an entire amino acidsequence of the reference sequence or any fragment specified asdescribed herein.

Such hGH polypeptide variants may be naturally occurring variants, suchas naturally occurring allelic variants encoded by one of severalalternate forms of a hGH occupying a given locus on a chromosome of anorganism, or isoforms encoded by naturally occurring splice variantsoriginating from a single primary transcript. Alternatively, a hGHpolypeptide variant may be a variant that is not known to occurnaturally and that can be made using art-known mutagenesis techniques.

It is known in the art that one or more amino acids may be deleted fromthe N-terminus or C-terminus of a bioactive peptide or protein withoutsubstantial loss of biological function (see for instance, Ron et al.,(1993), Biol Chem., 268 2984-2988 in which disclosure is herebyincorporated by reference in its entirety).

It also will be recognized by one of ordinary skill in the art that someamino acid sequences of hGH polypeptides can be varied withoutsignificant effect of the structure or function of the protein. Suchmutants include deletions, insertions, inversions, repeats, andsubstitutions selected according to general rules known in the art so asto have little effect on activity. For example, guidance concerning howto make phenotypically silent amino acid substitutions is provided inBowie et al. (1990), Science 247:1306-1310, hereby incorporated byreference in its entirety, wherein the authors indicate that there aretwo main approaches for studying the tolerance of an amino acid sequenceto change.

The first method relies on the process of evolution, in which mutationsare either accepted or rejected by natural selection. The secondapproach uses genetic engineering to introduce amino acid changes atspecific positions of a cloned hGH and selections or screens to identifysequences that maintain functionality. These studies have revealed thatproteins are surprisingly tolerant of amino acid substitutions. Theauthors further indicate which amino acid changes are likely to bepermissive at a certain position of the protein. For example, mostburied amino acid residues require nonpolar side chains, whereas fewfeatures of surface side chains are generally conserved. Other suchphenotypically silent substitutions are described in Bowie et al.,(1990) supra, and the references cited therein.

Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu and Pheinterchange of the hydroxyl residues Ser and Thr, exchange of the acidicresidues Asp and Glu, substitution between the amide residues Asn andGln, exchange of the basic residues Lys and Arg and replacements amongthe aromatic residues Phe, Tyr. In addition, the following groups ofamino acids generally represent equivalent changes: (1) Ala, Pro, Gly,Glu, Asp, Gln, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, Ile, Leu,Met, Ala, Phe; (4) Lys, Arg, His; (5) Phe, Tyr, Trp, His.

The term hGH polypeptide also encompasses all hGH polypeptides encodedby hGH analogs, orthologs, and/or species homologues. As used herein,the term “hGH analogs” refers to hGHs of different and unrelatedorganisms which perform the same functions in each organism but whichdid not originate from an ancestral structure that the organisms'ancestors had in common. Instead, analogous hGHs arose separately andthen later evolved to perform the same function (or similar functions).In other words, analogous hGH polypeptides are polypeptides with quitedifferent amino acid sequences but that perform the same biologicalactivity, namely promoting growth in the growing phase and inmaintaining normal body composition, anabolism, and lipid metabolism. Asused herein, the term “hGH orthologs” refers to hGHs within twodifferent species which sequences are related to each other via a commonhomologous hGH in an ancestral species but which have evolved to becomedifferent from each other. As used herein, the term “hGH homologs”refers to hGHs of different organisms which perform the same functionsin each organism and which originate from an ancestral structure thatthe organisms' ancestors had in common. In other words, homologous hGHpolypeptides are polypeptides with quite similar amino acid sequencesthat perform the same biological activity, namely promoting growth inthe growing phase and in maintaining normal body composition, anabolism,and lipid metabolism. Preferably, hGH polypeptide homologs may bedefined as polypeptides exhibiting at least 40%, 50%, 60%, 70%, 80%,90%, 95%, 96%, 97%, 98% or 99% identity to a reference hGH polypeptide,preferably the hGH polypeptide having a sequence as mentioned above.

Thus, a hGH polypeptide may be, for example: (i) one in which one ormore of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue mayor may not be oneencoded by the genetic code: or (ii) one in which one or more of theamino acid residues includes a substituent group: or (iii) one in whichthe hGH polypeptide is fused with another compound, such as a compoundto increase the half-life of the polypeptide (for example, polyethyleneglycol): or (iv) one in which the additional amino acids are fused tothe above form of the polypeptide, such as an IgG Fc fusion regionpeptide or leader or secretory sequence or a sequence which is employedfor purification of the above form of the polypeptide or a proproteinsequence.

hGH polypeptides may be monomers or multimers. Multimers may be dimers,trimers, tetramers or multimers comprising at least five monomericpolypeptide units. Multimers may also be homodimers or heterodimers.Multimers may be the result of hydrophobic, hydrophilic, ionic and/orcovalent associations and/or may be indirectly linked, by for example,liposome formation. In one example, covalent associations are betweenthe heterologous sequences contained in a fusion protein containing ahGH polypeptide or fragment thereof (see, e.g., U.S. Pat. No. 5,478,925,which disclosure is hereby incorporated by reference in its entirety).In another example, a hGH polypeptide or fragment thereof is joined toone or more polypeptides that may be either hGH polypeptides orheterologous polypeptides through peptide linkers such as thosedescribed in U.S. Pat. No. 5,073,627 (hereby incorporated by reference).

Another method for preparing multimer hGH polypeptides involves use ofhGH polypeptides fused to a leucine zipper or isoleucine zipperpolypeptide sequence known to promote multimerization of the proteins inwhich they are found using techniques known to those skilled in the artincluding the teachings of WO 94/10308. In another example, hGHpolypeptides may be associated by interactions between Flag® polypeptidesequence contained in fusion hGH polypeptides containing Flag®polypeptide sequence. hGH multimers may also be generated using chemicaltechniques known in the art such as cross-linking using linker moleculesand linker molecule length optimization techniques known in the art(see, e.g., U.S. Pat. No. 5,478,925), techniques known in the art toform one or more inter-molecule cross-links between the cysteineresidues located within the sequence of the polypeptides desired to becontained in the multimer (see, e.g., U.S. Pat. No. 5,478,925, additionof cysteine or biotin to the C terminus or N-terminus of hGH polypeptideand techniques to generate multimers containing one or more of thesemodified polypeptides (see, e.g., U.S. Pat. No. 5,478,925), or any ofthe 30 techniques to generate liposomes containing hGH multimers (see,e.g., U.S. Pat. No. 5,478,925,), which disclosures are incorporated byreference in their entireties.

As used herein, the term “hGH polypeptide fragment” refers to anypeptide or polypeptide comprising a contiguous span of a part of theamino acid sequence of an hGH polypeptide, preferably the polypeptidehaving the above-mentioned sequence.

rhGH PEGylated Prodrug—Preferred PEG, Polymer Chains

As discussed above, the rhGH PEGylated prodrug as described herein shallhave a relatively low activity.

Accordingly, in a preferred embodiment the total PEG load per growthhormone molecule amounts to at least 25 kDa. Generally the total PEGload will be less than 1000 kDa. Preferably, the PEG load is at least 25kDa and at most 500 kDa, even more preferably at least 30 kDa and atmost 250 kDa, even more preferably at least 30 kDa and at most 120 kDa,even more preferably at least 40 kDa and at most 100 kDa, even morepreferably at least 40 kDa and at most 90 kDa.

PEG may be attached to hGH through one or more anchoring points. In caseof one anchoring point, the corresponding PEG in the hGH PEG prodrugmonoconjugate will be branched and contain at least 3 chains. In case ofmore than one anchoring point, such as in a bisconjugate, thecorresponding PEG in the hGH PEG prodrug may be branched or linear.Bisconjugates may contain one or two transient linkages, and PEG may belinear or branched or contain a mixture of one linear and one branchedchain. In case the bisconjugate contains one transient linkage and onelinear and one branched chain the transient linkage may be on eitherchain. In case a branched PEG chain is used, there may be one or morebranching units.

A branched PEG is a PEG molecule consisting of a branching pointconnecting two or more PEG chains, to form a molecule with one anchoringpoint for attachment to growth hormone. This could be two 20 kDa PEGchains joined to form one branched 40 kDa PEG molecule. In the casewhere the molecule contains two or three branching points, the moleculeis referred to 3 and 4 armed PEG, respectively.

In summary and within the restrictions mentioned above, the PEG polymeris not limited to a particular structure and can be linear, branched, ormulti-armed (e.g. forked PEG or PEG attached to a polyol core),dendritic, or with degradable linkers.

Without being limited to theory the PEG load is intended to provide asuitable molecular mass to get the required relatively low activity andnot having a too high molecular mass of the PEG that could create otherproblems.

The PEGylation to native human GH may occur on several lysine groups oron the N-terminal amine (F1) as well described by Clark et al.(reference 2 herein) on page 21973 table III. Highly reactive arepositions F1 and LYS-140. Moderately reactive positions are LYS-115,LYS-38, and LYS-70. Poorly reactive are positions LYS-172, LYS-41,LYS-158 and LYS-168.

In more general terms the PEG used herein in combination with atransient linker may reduce the risk of lipoatrophy by suitable choiceof said polymer. However the principles of the present invention alsoapply to polymers other than PEG. Thus the term PEG is only used hereinexemplary for suitable polymers.

Thus, in a preferred embodiment, hGH PEG prodrug is a monoconjugateconjugated with one of its primary amino groups to an auto-cleavablefunctional group L^(a) to a polymer chain S⁰. This polymer chain S⁰ hasa molecular weight of at least 5 kDa and comprises at least onebranching structure BS¹. The branching structure BS¹ comprises a secondpolymer chain S¹, which has a molecular weight of at least 4 kDa.

As outlined above, at least a third polymer chain S² is required havinga molecular weight of at least 4 kDa. The polymer chain S² may be a partof BS¹ or may be a further branch of S⁰ or S¹ resulting in a furtherbranching structure BS², which comprises S².

Optionally more than 3 polymer chains are present in the prodrugconjugate of the present invention, e.g. 4, 5, 6, 7, or 8. However eachfurther polymer chain has a molecular weight of at least 4 kDa. Thetotal number of polymer chains is limited by the total weight of theprodrug conjugate being at most 1000 Da (without hGH-NH).

Thus a preferred embodiment of the present invention relates to acomposition, wherein at least one of the branching structures BS¹, BS²comprises a further fourth polymer chain S³ having a molecular weight ofat least 4 kDa or one of S⁰, S¹, S² comprises a third branchingstructure BS³ comprising the at least fourth polymer chain S³ having amolecular weight of at least 4 kDa.

The auto-cleavage inducing group G^(a), which is necessary for theauto-cleavage of L^(a) is comprised by one of the branching structuresor polymer chains.

Optionally, one of the branching structures serves as group G^(a) sothat the branching structure consists of G^(a) (instead of comprisingsaid group), which is also encompassed by the term “comprising”.

The preparation of a prodrug conjugate (AA) normally results in amixture of conjugates, where several primary amino groups of hGH areconjugated resulting in different mono-conjugated, differentbi-conjugated, different tri-conjugated, etc., prodrugs. Correspondingmonoconjugated, bisconjugated or trisconjugated hGH PEG prodrugs can beseparated by standard methods known in the art, like columnchromatography and the like.

In monoconjugates of hGH PEG prodrugs, the at least three polymer chainsS⁰, S¹, S² contain a “polymer moiety”, which is characterized by one ormore repeating units, which may be randomly, block wise or alternatingdistributed. In addition, the at least three polymer chains S⁰, S¹, S²show an end group, which is typically a hydrogen atom or an alkyl grouphaving from 1 to 6 carbon atoms, which may be branched or unbranched,e.g. a methyl group, especially for PEG based polymer chains resultingin so called mPEGs.

It is pointed out that the polymer moieties within the at least threepolymer chains S⁰, S¹, S² may have further chain-like substituents,originating from the repeating units and resulting in chains having lessthan 4 kDa of molecular weight and which are not considered as polymerchains S⁰, S¹, S², etc. Preferably, the at least three polymer chainsS⁰, S¹, S² carry substituents of less than 1000 Da molecular weight.

A relevant structural feature of S⁰ is its critical distance. Thecritical distance defines the shortest distance between the attachmentsite of S⁰ to L^(a) and the first branching structure BS¹ measured asconnected atoms. The length of the critical distance has an effect onthe residual activity as discussed for compound 33. The criticaldistance is preferably less than 50, more preferred less than 20, mostpreferred less than 10.

The at least three polymer chains S⁰, S¹ and S² typically each containan interconnecting moiety. Ga is present in at least one of theinterconnecting moieties. For polymer chains other than S⁰, theinterconnecting moiety is the structural element connecting the polymermoiety of for instance S¹ with BS¹ and the polymer moiety of S² withBS². For S⁰, the interconnecting moiety is the structural elementconnecting L^(a) and BS¹.

Interconnecting moieties may consist of a C₁₋₅₀ alkyl chain, which isbranched or unbranched and which is optionally interrupted or terminatedby hetero atoms or functional groups selected from the group consistingof —O—; —S—; N(R); C(O); C(O)N(R); N(R)C(O); one or more carbocycles orheterocycles, wherein R is hydrogen or a C₁₋₂₀ alkyl chain, which isoptionally interrupted or terminated by one or more of theabovementioned atoms or groups, which further have a hydrogen asterminal atom; and wherein a carbocycle is phenyl; naphthyl; indenyl;indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; and wherein the heterocycle is a4 to 7 membered heterocyclyl; or 9 to 11 membered heterobicyclyl.

“C₃₋₁₀ cycloalkyl” or “C₃₋₁₀ cycloalkyl ring” means a cyclic alkyl chainhaving 3 to 10 carbon atoms, which may have carbon-carbon double bondsbeing at least partially saturated, e.g. cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl,cyclononyl, cyclodecyl. Each hydrogen of a cycloalkyl carbon may bereplaced by a substituent. The term “C₃₋₁₀ cycloalkyl” or “C₃₋₁₀cycloalkyl ring” also includes bridged bicycles like norbonane ornorbonene.

“4 to 7 membered heterocyclyl” or “4 to 7 membered heterocycle” means aring with 4, 5, 6 or 7 ring atoms that may contain up to the maximumnumber of double bonds (aromatic or non-aromatic ring which is fully,partially or un-saturated) wherein at least one ring atom up to 4 ringatoms are replaced by a heteroatom selected from the group consisting ofsulfur (including —S(O)—, —S(O)₂—), oxygen and nitrogen (including═N(O)—) and wherein the ring is linked to the rest of the molecule via acarbon or nitrogen atom. Examples for a 4 to 7 membered heterocycles areazetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrroline,imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline,isoxazole, isoxazoline, thiazole, thiazoline, isothiazole,isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran,tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine,oxazolidine, isoxazolidine, thiazolidine, isothiazolidine,thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran,imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine,piperidine, morpholine, tetrazole, triazole, triazolidine,tetrazolidine, diazepane, azepine or homopiperazine.

“9 to 11 membered heterobicyclyl” or “9 to 11 membered heterobicycle”means a heterocyclic system of two rings with 9 to 11 ring atoms, whereat least one ring atom is shared by both rings and that may contain upto the maximum number of double bonds (aromatic or non-aromatic ringwhich is fully, partially or un-saturated) wherein at least one ringatom up to 6 ring atoms are replaced by a heteroatom selected from thegroup consisting of sulfur (including —S(O)—, —S(O)₂—), oxygen andnitrogen (including ═N(O)—) and wherein the ring is linked to the restof the molecule via a carbon or nitrogen atom. Examples for a 9 to 11membered heterobicycle are indole, indoline, benzofuran, benzothiophene,benzoxazole, benzisoxazole, benzothiazole, benzisothiazole,benzimidazole, benzimidazoline, quinoline, quinazoline,dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline,decahydroquinoline, isoquinoline, decahydroisoquinoline,tetrahydroisoquinoline, dihydroisoquinoline, benzazepine, purine orpteridine. The term 9 to 11 membered heterobicycle also includes spirostructures of two rings like 1,4-dioxa-8-azaspiro[4.5]decane or bridgedheterocycles like 8-aza-bicyclo[3.2.1]octane.

The carbocycle, heterocycle and heterobicycle may be substituted byC₁₋₂₀ alkyl, optionally interrupted or terminated by hetero atoms orfunctional groups selected from the group consisting of —O—; —S—; N(R);C(O); C(O)N(R); N(R)C(O), wherein R is hydrogen or a C₁₋₁₀ alkyl chain,which is optionally interrupted or terminated by one or more of theabovementioned atoms or groups, which further have a hydrogen asterminal atom.

The polymer moiety of the at least three chains S⁰, S¹, S² form themajority part of the chains, preferably at least 90% of the molecularweight of each chain, more preferred at least 95%, even more preferredat least 97.5%, even more preferred at least 99%. Thus, the basis of thechains is represented by the polymer moiety.

Preferably, the at least three chains S⁰, S¹, S² are independently basedon a polymer selected from the group consisting of polyalkyloxypolymers, hyaluronic acid and derivatives thereof, polyvinyl alcohols,polyoxazolines, polyanhydrides, poly(ortho esters), polycarbonates,polyurethanes, polyacrylic acids, polyacrylamides, polyacrylates,polymethacrylates, polyorganophosphazenes, polysiloxanes,polyvinylpyrrolidone, polycyanoacrylates, and polyesters.

Preferably, the at least three chains S⁰, S¹, S² are based on the samepolymer. Preferably, the at least three chains S⁰, S¹, S² are based onpolyalkyoxy polymers. Even more preferred the at least three chains S⁰,S¹, S² are polyethylene glycol based.

The same applies for further chains S³, S⁴, S⁵, etc, accordingly.

The chain S⁰ comprises a branching structure BS¹, so that S¹ is linkedto S⁰. For the linkage of S² the branching structure BS¹ may be used ora further branching structure BS² is present, which may be a part S⁰ orS¹. Accordingly, further branching structures may be present, whenfurther chains are present. For example in case a chain S³ is present itmay be linked to BS¹, BS² or a branching structure BS³. The branchingstructure BS³, if present, may be part of S⁰, S¹, or S².

In general any chemical entity, which allows the branching of a chain,may be used. Preferably, the branching structures are independentlyselected from the group consisting of at least 3-fold substitutedcarbocycle, at least 3-fold substituted heterocycle, a tertiary carbonatom, a quaternary carbon atom, and a tertiary nitrogen atom, whereinthe terms carbocycle and heterocycle are defined as indicated above.

rhGH PEGylated Prodrug—Transient Linker Structures, L^(a), G^(a)

In publications in the field of auto-cleavage inducing groups aresometimes called linkers to discriminate their structure from thecarrier. Nevertheless it is often difficult to clearly separate thesestructural features. Therefore, within the meaning of the presentinvention the cleavage-inducing group G^(a) is considered to be part ofthe carrier S, comprising at least S⁰, S¹, S², BS¹, and optionally BS².Variation of the chemical nature of G^(a) allows the engineering of theproperties of the self-cleaving properties of a correspondingcarrier-linked prodrug to a great extent.

As discussed above, a PEGylated-prodrug, wherein the drug is for examplerhGH as described in patent application WO-A 2005/099768, and has acharacteristic of release, which is therein described as the 1,6cleavage system without the production of toxic aromatic compounds. Inthis document is broadly described numerous herein relevant suitabletransient linker structures to get a relevant release profile ofinterest.

Other transient linker structures are generically/broadly described ine.g. other Complex Biosystems GmbH applications such as WO-A2005/034909, WO-A 2005/099768, WO-A 2006/003014 and WO-A 2006/136586.

More transient linker structures are broadly described in e.g. WO-A99/30727 (Enzon Inc).

In order to solve the present problems for GH as discussed herein, thepresent inventors have selected suitable preferred transient linkerstructures to get the herein described relevant functional properties ofthe rhGH PEGylated prodrug. Based on the herein detailed description ofpreferred linker structures it is within the skilled person knowledge tomake other suitable preferred transient linker structures that couldgive an rhGH PEGylated prodrug with the herein described relevantfunctional properties.

Especially, suitable transient linker structures, which are selfhydrolysable (auto-cleavable) can be chosen for incorporation into S⁰.The herein selected linker structures are described in detail below.

Ideally, a conjugate of the invention will possess one or more of thefollowing features and/or advantages over current rhGH conjugates orformulations; can easily be synthesized in good yields, have half life'sfalling within preferred range, can be purified to provide homogeneousconjugate compositions, exhibit activity after autocleavage such as invitro and in vivo activity and have pharmacodynamic effects superior tounmodified rhGH and previously described rhGH conjugates and do notcause lipoatrophy. The herein described structures exhibit releaseproperties as required herein.

In general, carrier-linked prodrugs require the presence of a cleavablefunctional group connecting drug and carrier. In the absence ofautohydrolysis-inducing groups, functional groups that involve adrug-donated amino group such as aliphatic amide or carbamate bondsL^(a) are usually very stable against hydrolysis and the rate ofcleavage of the amide bond would be too slow for therapeutic utility ina prodrug system.

If such stable linkages are to be used in carrier-linked prodrugs,cleavage of the functional group is not possible in a therapeuticallyuseful timeframe without biotransformation. In these cases, the linkerdisplays a structural motif that is recognized as a substrate by acorresponding endogenous enzyme. In such a case, the cleavage of thefunctional bond L^(a) involves a complex comprising the enzyme. Examplesare peptide linkers that are recognized by endogenous proteases andcleaved enzymatically.

Enzyme levels may differ significantly between individuals resulting inbiological variation of prodrug activation by the enzymatic cleavage.The enzyme levels may also vary depending on the site of administration.For instance it is known that in the case of subcutaneous injection,certain areas of the body yield more predictable therapeutic effectsthan others. Such high level of interpatient variability is notdesirable. Furthermore, it is difficult to establish an in vivo-in vitrocorrelation of the pharmacokinetic properties for such enzyme-dependentcarrier-linked prodrugs. In the absence of a reliable in vivo-in vitrocorrelation optimization of a release profile becomes a cumbersome task.

In order to avoid patient-to-patient and injection site variability, itis desirable to employ carrier-linked prodrugs that exhibit cleavagekinetics in a therapeutically useful timeframe without the requirementfor additional enzymatic contribution to cleavage. Especially for highmolecular weight carriers, specifically for branched polymeric carriers,access to the connecting functional group La may be restricted forenzymes due to sterical crowding. Therefore there exists a need todevise carrier-linked prodrugs that exhibit self-cleaving properties.

Autocleavage kinetics can for instance be measured in vitro by recordinghydrolysis rates in buffered solution without enzyme.

In order to introduce hydrolytic lability into functional groups L suchas amides or carbamates, it is necessary to engineer structural chemicalcomponents into the carrier in order to function for instance asneighbouring groups in proximity to the functional group. Suchautocleavage-inducing chemical structures that exert control over thecleavability of the prodrug amide bond are termed auto-cleavage inducinggroups G^(a). Autocleavage-inducing groups can have a strong effect onthe rate of hydrolysis of a given functional group L^(a).

Preferred L^(a) are selected from the group consisting of C(O)—O—, andC(O)—, which form together with the primary amino group of hGH acarbamate or amide group.

Thus, a composition of the present invention is preferred, wherein L^(a)is selected from the group consisting of C(O)—O—, and C(O)—, which formtogether with the primary amino group of hGH a carbamate or amide groupresulting in formula (AA1) or (AA2)

hGH-NH—C(O)O—S⁰   (AA1),

hGH-NH—C(O)—S⁰   (AA2).

The following sections will list various structural components that mayfunction as cleavage-inducing groups G^(a).

The group G^(a) represents an autocleavage inducing group. G^(a) may bepresent as such or as a cascade autocleavage-inducing group, which isunmasked to become effective by means of an additional hydrolytical orenzymatic cleavage step. If G^(a) is present as such, it governs therate-limiting autohydrolysis of L^(a).

Examples for G^(a):

A. J. Garman et al. (A. J. Garman, S. B. Kalindjan, FEBS Lett. 1987, 223(2), 361-365 1987) used PEG5000-maleic anhydride for the reversiblemodification of amino groups in tissue-type plasminogen activator andurokinase. Regeneration of functional enzyme from PEG-uPA conjugate uponincubation at pH 7.4 buffer by cleavage of the maleamic acid linkagefollowed first order kinetics with a half-life of 6.1 h.

Simple aromatic moieties may infer lability to a connected carbamatebond (WO-A 01/47562). For instance, substituted or unsubstitutedfluorenylmethyl group were used to labilize carbamate linkages tovarious bioactive agents in a prodrug approach (Tsubery et al. J BiolChem 279 (2004) 38118-24). Two PEG chains were attached to a fluorenylmoiety in WO-A 2007/075534.

Thus, G^(a) is an aromatic ring or fluorenylmethyl directly attached toa carbamate functional group L^(a).

Accordingly, a composition of the present invention is preferred,wherein G^(a) is an aromatic ring or fluorenylmethyl directly attachedto a carbamate functional group formed by L^(a) and the primary aminogroup of hGH.

Alternatively, transformation of G^(a) may induce a molecularrearrangement within S⁰ such as a 1,4 or 1,6-elimination. Therearrangement renders L^(a) so much more labile that its cleavage isinduced. The transformation of G^(a) is the rate-limiting step in thecascade mechanism. Ideally, the cleavage rate of the temporary linkageis identical to the desired release rate for the drug molecule in agiven therapeutic scenario. In such a cascade system base on 1,6elimination, it is desirable that the cleavage of L^(a) is substantiallyinstantaneous after its lability has been induced by transformation ofG^(a). In addition it is desirable that the rate-limiting cleavagekinetics proceed in a therapeutically useful timeframe without therequirement for additional enzymatic contribution in order to avoid thedrawbacks associated with predominantly enzymatic cleavage discussedabove.

R. B. Greenwald, A. Pendri, C. D. Conover, H. Zhao, Y. H. Choe, A.Martinez, K. Shum, S. Guan, J. Med. Chem., 1999, 42, 3657-3667 & PCTPatent Application WO-A 99/30727 described a methodology forsynthesizing poly(ethylene glycol) prodrugs of amino-containing smallmolecule compounds based on 1,4- or 1,6-benzyl elimination. In thisapproach the amino group of the drug molecule is linked via a carbamategroup to a PEGylated benzyl moiety. The poly(ethylene glycol) wasattached to the benzyl group by ester, carbonate, carbamate, or amidebonds. The release of PEG from the drug molecule occurs through acombination of autohydrolysis and enzymatic cleavage. The cleavage ofthe release-triggering masking group is followed in this approach by theclassical and rapid 1,4- or 1,6-benzyl elimination. This linker systemwas also used for releasable poly(ethylene glycol) conjugates ofproteins (S. Lee, R. B. Greenwald et al. Bioconj. Chem. 2001, 12 (2),163-169). Lysozyme was used as model protein because it loses itsactivity when PEGylation takes place on the epsilon-amino group oflysine residues. Various amounts of PEG linker were conjugated to theprotein. Regeneration of native protein from the PEG conjugates occurredin rat plasma or in non-physiological high pH buffer. See also F. M. H.DeGroot et al. (WO-A 2002/083180 and WO-A 2004/043493), and D. Shabat etal. (WO-A 2004/019993).

Thus, L^(a) is a carbamate functional group, the cleavage of said groupis induced by a hydroxyl or amino group of G^(a) via 1,4- or 1,6 benzylelimination of S⁰, wherein G^(a) contains ester, carbonate, carbamate,or amide bonds that undergo rate-limiting transformation. In effect,G^(a) may be cleaved off by hydrolysis.

Accordingly, a composition of the present invention is preferred,wherein L^(a) forms together with the amino group of hGH a carbamatefunctional group, the cleavage of said group is induced by a hydroxyl oramino group of G^(a) via 1,4- or 1,6 benzyl elimination of S⁰, whereinG^(a) contains ester, carbonate, carbamate, or amide bonds that undergorate-limiting transformation.

G^(a) may contain a cascade cleavage system that is enabled bycomponents of Ga that are composed of a structural combinationrepresenting the aforementioned precursor. A precursor of G^(a) maycontain additional temporary linkages such as an amide, ester or acarbamate. The stability, or susceptibility to hydrolysis of theprecursor's temporary linkage (e.g. carbamate) may be governed byautohydrolytic properties or may require the activity of an enzyme.

Antczak et al. (Bioorg Med Chem 9 (2001) 2843-48) describe a reagentwhich forms the basis for a macromolecular cascade prodrug system foramine-containing drug molecules. In this approach an antibody serves asthe carrier, a stable bond connects the antibody to an activating group,carrying a cleavable masking group. Upon removal of the ester-linkedmasking group, La cleaves and releases the drug compound.

D. Shabat et al. (Chem. Eur. 3. 2004, 10, 2626-2634) describe apolymeric prodrug system based on a mandelic acid activating group. Inthis system the masking group is linked to the activating group by acarbamate bond. The activating group is conjugated permanently to apolyacrylamide polymer via an amide bond. After activation of themasking group by a catalytic antibody, the masking group is cleaved bycyclization and the drug is released. The activating group is stillconnected to the polyacrylamide polymer after drug release.

M.-R. Lee at al. describe (Angew. Chem. 2004, 116, 1707-17 10) a similarprodrug system based on mandelic acid activating group and ester-linkedmasking group.

Nevertheless in these linkers a 1,6 elimination step still generates ahighly reactive aromatic intermediate. Even if the aromatic moietyremains permanently attached to the polymeric carrier, side reactionswith potentially toxic or immunogenic effects may be caused.

Greenwald et al. published in 2000 a poly(ethylene glycol) drug deliverysystem of amino-containing prodrugs based on trimethyl locklactonization (R. B. Greenwald et al. J. Med. Chem. 2000, 43(3),457-487; WO-A 02/089789). In this prodrug system substitutedo-hydroxyphenyl-dimethylpropionic acid is coupled to amino groups ofdrug molecules by an amide bond. The hydroxy group is linked to PEG byan ester, carbonate, or carbamate group. The rate determining step indrug release is the enzymatic cleavage of these functional groupsfollowed by fast amide cleavage by lactonization, liberating an aromaticlactone side product.

More recently, R. B. Greenwald et al. (Greenwald et al. J. Med. Chem.2004, 47, 726-734) described a PEG prodrug system based onbis-(N-2-hydroxyethyl)glycin amide (bicin amide) linker. In this systemtwo PEG molecules are linked to a bicin molecule coupled to an aminogroup of the drug molecule. The first two steps in prodrug activation isthe enzymatic cleavage of both PEG molecules. Different linkages betweenPEG and bicin are described resulting in different prodrug activationkinetics. The main disadvantage of this system is the slow hydrolysisrate of bicin amide conjugated to the drug molecule (t_(1/2)=3 h inphosphate buffer) resulting in the release of a bicin-modified prodrugintermediate that may show different pharmacokinetic and pharmacodynamicproperties than the parent drug molecule.

More specifically, preferred groups L^(a) and G^(a) with specific spacermoieties for S⁰ are described below.

A preferred structure according to WO-A 2005/099768 is selected from thegeneral formula (I) and (II):

wherein T represents hGH-NH; X represents a spacer moiety; Y₁ and Y₂each independently represent O, S or NR₆; Y₃ represents O or S; Y₄represents O, NR₆ or —C(R₇)(R₈); R₃ represents a moiety selected fromthe group consisting of hydrogen, substituted or unsubstituted linear,branched or cyclical alkyl or heteroalkyl groups, aryls, substitutedaryls, substituted or unsubstituted heteroaryls, cyano groups, nitrogroups, halogens, carboxy groups, carboxyalkyl groups, alkylcarbonylgroups or carboxamidoalkyl groups; R₄ represents a moiety selected fromthe group consisting of hydrogen, substituted or unsubstituted linear,branched or cyclical alkyls or heteroalkyls, aryls, substituted aryls,substituted or unsubstituted heteroaryl, substituted or unsubstitutedlinear, branched or cyclical alkoxys, substituted or unsubstitutedlinear, branched or cyclical heteroalkyloxys, aryloxys orheteroaryloxys, cyano groups and halogens; R₇ and R₈ are eachindependently selected from the group consisting of hydrogen,substituted or unsubstituted linear, branched or cyclical alkyls orheteroalkyls, aryls, substituted aryls, substituted or unsubstitutedheteroaryls, carboxyalkyl groups, alkylcarbonyl groups, carboxamidoalkylgroups, cyano groups, and halogens; R₆ represents a group selected fromhydrogen, substituted or unsubstituted linear, branched or cyclicalalkyls or heteroalkyls, aryls, substituted aryls and substituted orunsubstituted heteroaryls; R₁ represents the rest of S⁰; W represents agroup selected from substituted or unsubstituted linear, branched orcyclical alkyls, aryls, substituted aryls, substituted or unsubstitutedlinear, branched or cyclical heteroalkyls, substituted or unsubstitutedheteroaryls; Nu represents a nucleophile; n represents zero or apositive imager; and Ar represents a multi-substituted aromatichydrocarbon or multi-substituted aromatic heterocycle.

Within the meaning of the present invention, the group L^(a) isrepresented by Y₃—C(Y₅)NH— (together with the amino group of hGH), G^(a)is represented by Nu-W—Y₄—C(Y₁)Y₂ and Ar(R₄)_(n)—C(R₃)XR₁ represents S⁰,which further includes at least S¹, S², BS¹ and optionally BS².

In an alternative embodiment S¹ is attached via Ar or represents R₃.Then the carbon atom adjacent to Y₃ substituted with XR¹ represents thebranching structure BS¹, S¹ is terminated with Ar comprising G^(a). Itis evident that in this embodiment terms S⁰ and S¹ are interchangeable.

Preferably, in formula (AA) or (AA1) S⁰ is of formula (AAA1)

wherein

G^(a) has the meaning as indicated above;

S⁰⁰ is CH₂; or C(O);

S^(0A) is an alkylene chain having from 1 to 20 carbon atoms, which isoptionally interrupted or terminated by one or more groups, cycles orheteroatoms selected from the group consisting of optionally substitutedheterocycle; O; S; C(O); and NH;

BS¹, BS², BS³ are independently selected from the group consisting of N;and CH.

S^(0B), S^(1A) are independently an alkylene chain having from 1 to 200carbon atoms, which is optionally interrupted or terminated by one ormore groups, cycles or heteroatoms selected from the group consisting ofoptionally substituted heterocycle; O; S; C(O); and NH;

S^(0C), S^(1B), are (C(O))_(n2)(CH₂)_(n1)(OCH₂CH₂)_(n)OCH₃, wherein eachn is independently an integer from 100 to 500, each n1 is independently0, 1, 2, 3, 4, 5, 6, 7,or 8, and n2 is 0 or 1.

S², S³ are independently hydrogen; or(C(O))_(n2)(CH₂)_(n1)(OCH₂CH₂)_(n)OCH₃, wherein each n is independentlyan integer from 100 to 500, each n1 is independently 0, 1, 2, 3, 4, 5,6, 7,or 8, and n2 is 0 or 1, provided that at least one of S², S³ isother than hydrogen;

R², R³ are defined as for formula (A) below.

The term heterocycle means an heterocycle as defined above. Optionalsubstituents are, e.g. oxo (═O), where the ring is at least partiallysaturated, a branched or unbranched alkyl chain having from one to 6carbon atoms, or halogen. A preferred substituted heterocycle issuccinimide.

Preferably, G^(a) in formula (AAA1) is OC(O)—R and R is the partialstructure of formula (I) as shown below, wherein R1, R4, R5 and n aredefined as given below.

Another preferred embodiment is described in WO06136586A2. Accordingly,the following structures are preferred:

wherein T is NH-hGH;

X is a spacer moiety such as R13-Y1;

Y1 is O, S, NR6, succinimide, maleimide, unsaturated carbon-carbon bondsor any heteratom containing a free electron pair or is absent;

R13 is selected from substituted or non-substituted linear, branched orcyclical alkyl or heteroalkyl, aryls, substituted aryls, substituted ornon-substituted heteroaryls;

R2 and R3 are selected independently from hydrogen, acyl groups, orprotecting groups for hydroxyl groups;

R4 to R12 are selected independently from hydrogen, X—R1, substituted ornon-substituted linear, branched or cyclical alkyl or heteroalkyl,aryls, substituted aryls, substituted or non-substituted heteroaryls,cyano, nitro, halogen, carboxy, carboxamide;

R1 is the rest of S⁰, comprising at least S¹, S², BS¹, and optionallyBS².

In this embodiment L^(a) is an amide group, and G^(a) encompasses theN-branched structure carrying OR₂/OR₃.

In yet another preferred embodiment, a preferred structure is given by aprodrug conjugate D-L, wherein

-D is NH-hGH; and

-L is a

non-biologically active linker moiety -L¹ represented by formula (I),

wherein the dashed line indicates the attachment to the amino group ofhGH by forming an amide bond;

X is C(R⁴R^(4a)); N(R⁴); O; C(R⁴R^(4a))—C(R⁵R^(5a));C(R⁵R^(5a))—C(R⁴R^(4a)); C(R⁴R^(4a))—N(R⁶); N(R⁶)—C(R⁴R^(4a));C(R⁴R^(4a))—O; or O—C(R⁴R^(4a));

X¹ is C; or S(O);

X² is C(R⁷, R^(7a)); or C(R⁷, R^(7a))—C(R⁸, R^(8a));

R¹, R^(1a), R², R^(2a), R³, R^(3a), R⁴, R^(4a), R⁵, R^(5a), R⁶, R⁷,R^(7a), R⁸, R^(8a) are independently selected from the group consistingof H; and C₁₋₄ alkyl; or

Optionally, one or more of the pairs R^(1a)/R^(4a), R^(1a)/R^(5a),R^(4a)/R^(5a), R^(4a)/R^(5a), R^(7a)/R^(8a) form a chemical bond;

Optionally, one or more of the pairs R¹/R^(1a), R²/R^(2a), R⁴/R^(4a),R⁵/R^(5a), R⁷/R^(7a), R⁸/R^(8a) are joined together with the atom towhich they are attached to form a C₃₋₇ cycloalkyl; or 4 to 7 memberedheterocyclyl;

Optionally, one or more of the pairs R¹/R⁴, R¹/R⁵, R¹/R⁶, R⁴/R⁵, R⁷/R⁸,R²/R³ are joined together with the atoms to which they are attached toform a ring A;

Optionally, R³/R^(3a) are joined together with the nitrogen atom towhich they are attached to form a 4 to 7 membered heterocycle;

A is selected from the group consisting of phenyl; naphthyl; indenyl;indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; 4 to 7 membered heterocyclyl; and9 to 11 membered heterobicyclyl; and

wherein L¹ is substituted with one group L²-Z and optionally furthersubstituted, provided that the hydrogen marked with the asterisk informula (I) is not replaced by a substituent; wherein

L² is a single chemical bond or a spacer; and

Z is the rest of S⁰, comprising at least S¹, S², BS¹, and optionallyBS².

In this embodiment L^(a) is represented by an amide group and G^(a) isrepresented by N(H*)X¹(O) and the chain connecting to N includingsubtituents of N.

Prodrug conjugates of this type are described in European Patentapplication N^(o) 08150973.9

Accordingly, a composition of the present invention is preferred,wherein L^(a)-S⁰ is represented by formula (AAA2),

wherein the dashed line indicates the attachment to the primary aminogroup of hGH so that L^(a) and the amino group form an amide bond;

X is C(R⁴R^(4a)); N(R⁴); O; C(R⁴R^(4a))—C(R⁵R^(5a));C(R⁵R^(5a))—C(R⁴R^(4a)); C(R⁴R^(4a))—N(R⁶); N(R⁶)—C(R⁴R^(4a));C(R⁴R^(4a))—O; or O—C(R⁴R^(4a));

X¹ is C; or S(O);

X² is C(R⁷, R^(7a)); or C(R⁷, R^(7a))—C(R⁸, R^(8a));

R¹, R^(1a), R², R^(2a), R³, R^(3a), R⁴, R^(4a), R⁵, R^(5a), R⁶, R⁷, R⁸,R^(8a) are independently selected from the group consisting of H; andC₁₋₄ alkyl; or

Optionally, one or more of the pairs R^(1a)/R^(4a), R^(1a)/R^(5a),R^(4a)/R^(5a), R^(4a)/R^(5a), R^(7a)/R^(8a) form a chemical bond;

Optionally, one or more of the pairs R¹/R^(1a), R²/R^(2a), R⁴/R^(4a),R⁵/R^(5a), R⁷/R^(7a), R⁸/R^(8a) are joined together with the atom towhich they are attached to form a C₃₋₇ cycloalkyl; or 4 to 7 memberedheterocyclyl;

Optionally, one or more of the pairs R¹/R⁴, R¹/R⁵, R¹/R⁶, R⁴/R⁵, R⁷/R⁸,R²/R³ are joined together with the atoms to which they are attached toform a ring A;

Optionally, R³/R^(3a) are joined together with the nitrogen atom towhich they are attached to form a 4 to 7 membered heterocycle;

A is selected from the group consisting of phenyl; naphthyl; indenyl;indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; 4 to 7 membered heterocyclyl; and9 to 11 membered heterobicyclyl; and

wherein S⁰ is substituted with one group L²-Z and optionally furthersubstituted, provided that the hydrogen marked with the asterisk informula (I) is not replaced by a substituent; wherein

L² is a single chemical bond or a spacer; and

Z is of formula (AAA2a)

wherein S⁰⁰, S^(0A), S^(0B), S^(0C), S^(1A), S^(1B), S², S³, BS¹, BS²,and BS³ have the meaning as indicated for formula (AAA1) above.

“Alkyl” means a straight-chain or branched carbon chain. Each hydrogenof an alkyl carbon may be replaced by a substituent.

“C₁₋₄ alkyl” means an alkyl chain having 1-4 carbon atoms, e.g. ifpresent at the end of a molecule: methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl tert-butyl, or e.g. —CH₂—, —CH₂—CH₂—,—CH(CH₃)—, —CH₂—CH₂—CH₂—, —CH(C₂H₅)—, —C(CH₃)₂—, when two moieties of amolecule are linked by the alkyl group. Each hydrogen of a C₁₋₄ alkylcarbon may be replaced by a substituent.

“C₁₋₆ alkyl” means an alkyl chain having 1-6 carbon atoms, e.g. ifpresent at the end of a molecule: C₁₋₄ alkyl, methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl; tert-butyl, n-pentyl, n-hexyl,or e.g. —CH₂—, —CH₂—CH₂—, —CH(CH₃)—, —CH₂—CH₂—CH₂—, —CH(C₂H₅)—,—C(CH₃)₂—, when two moieties of a molecule are linked by the alkylgroup. Each hydrogen of a C₁₋₆ alkyl carbon may be replaced by asubstituent.

Accordingly, “C₁₋₁₈ alkyl” means an alkyl chain having 1 to 18 carbonatoms and “C₈₋₁₈ alkyl” means an alkyl chain having 8 to 18 carbonatoms. Accordingly, “C₁₋₅₀ alkyl” means an alkyl chain having 1 to 50carbon atoms.

“C₂₋₅₀ alkenyl” means a branched or unbranched alkenyl chain having 2 to50 carbon atoms, e.g. if present at the end of a molecule: —CH═CH₂,—CH═CH—CH₃, —CH₂—CH═CH₂, —CH═CH—CH₂—CH₃, —CH═CH—CH═CH₂, or e.g. —CH═CH—,when two moieties of a molecule are linked by the alkenyl group. Eachhydrogen of a C₂₋₅₀ alkenyl carbon may be replaced by a substituent asfurther specified. Accordingly, the term “alkenyl” relates to a carbonchain with at least one carbon carbon double bond. Optionally, one ormore triple bonds may occur.

“C₂₋₅₀ alkynyl” means a branched or unbranched alkynyl chain having 2 to50 carbon atoms, e.g. if present at the end of a molecule: —C≡CH,—CH₂—C≡CH, CH₂—CH₂—C≡CH, CH₂—C≡C—CH₃, or e.g. —C≡C— when two moieties ofa molecule are linked by the alkynyl group. Each hydrogen of a C₂₋₅₀alkynyl carbon may be replaced by a substituent as further specified.Accordingly, the term “alkynyl” relates to a carbon chain with at lestone carbon carbon triple bond. Optionally, one or more double bonds mayoccur.

“C₃₋₇ cycloalkyl” or “C₃₋₇ cycloalkyl ring” means a cyclic alkyl chainhaving 3 to 7 carbon atoms, which may have carbon-carbon double bondsbeing at least partially saturated, e.g. cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl. Each hydrogen of acycloalkyl carbon may be replaced by a substituent. The term “C₃₋₇cycloalkyl” or “C₃₋₇ cycloalkyl ring” also includes bridged bicycleslike norbonane or norbonene. Accordingly, “C₃₋₅ cycloalkyl” means acycloalkyl having 3 to 5 carbon atoms.

Accordingly, “C₃₋₁₀ cycloalkyl” means a cyclic alkyl having 3 to 10carbon atoms, e.g. C₃₋₇ cycloalkyl; cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl,cyclononyl, cyclodecyl. The term “C₃₋₁₀ cycloalkyl” also includes atleast partially saturated carbomono- and -bicycles.

“Halogen” means fluoro, chloro, bromo or iodo. It is generally preferredthat halogen is fluoro or chloro.

“4 to 7 membered heterocyclyl” or “4 to 7 membered heterocycle” means aring with 4, 5, 6 or 7 ring atoms that may contain up to the maximumnumber of double bonds (aromatic or non-aromatic ring which is fully,partially or un-saturated) wherein at least one ring atom up to 4 ringatoms are replaced by a heteroatom selected from the group consisting ofsulfur (including —S(O)—, —S(O)₂—), oxygen and nitrogen (including═N(O)—) and wherein the ring is linked to the rest of the molecule via acarbon or nitrogen atom. Examples for a 4 to 7 membered heterocycles areazetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrroline,imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline,isoxazole, isoxazoline, thiazole, thiazoline, isothiazole,isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran,tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine,oxazolidine, isoxazolidine, thiazolidine, isothiazolidine,thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran,imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine,piperidine, morpholine, tetrazole, triazole, triazolidine,tetrazolidine, diazepane, azepine or homopiperazine.

“9 to 11 membered heterobicyclyl” or “9 to 11 membered heterobicycle”means a heterocyclic system of two rings with 9 to 11 ring atoms, whereat least one ring atom is shared by both rings and that may contain upto the maximum number of double bonds (aromatic or non-aromatic ringwhich is fully, partially or un-saturated) wherein at least one ringatom up to 6 ring atoms are replaced by a heteroatom selected from thegroup consisting of sulfur (including —S(O)—, —S(O)₂—), oxygen andnitrogen (including ═N(O)—) and wherein the ring is linked to the restof the molecule via a carbon or nitrogen atom. Examples for a 9 to 11membered heterobicycle are indole, indoline, benzofuran, benzothiophene,benzoxazole, benzisoxazole, benzothiazole, benzisothiazole,benzimidazole, benzimidazoline, quinoline, quinazoline,dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline,decahydroquinoline, isoquinoline, decahydroisoquinoline,tetrahydroisoquinoline, dihydroisoquinoline, benzazepine, purine orpteridine. The term 9 to 11 membered heterobicycle also includes spirostructures of two rings like 1,4-dioxa-8-azaspiro[4.5]decane or bridgedheterocycles like 8-aza-bicyclo[3.2.1]octane.

Preferably, L^(a)-S⁰ is selected from the group consisting of

wherein R is H; or C₁₋₄ alkyl; Y is NH; O; or S; and R¹, R^(1a), R²,R^(2a), R³, R^(3a), R⁴, X, X¹, X² have the meaning as indicated above.

Even more preferred, L^(a)-S⁰ is selected from the group consisting of

wherein R has the meaning as indicated above.

At least one (up to four) hydrogen is replaced by a group L²-Z. In casemore than one group L²-Z is present each L² and each Z can be selectedindependently. Preferably, only one group L²-Z is present.

In general, S⁰ can be substituted with L²-Z at any position apart fromthe replacement of the hydrogen marked with an asterisk in the formulaeabove. Preferably, one to four of the hydrogen given by R, R¹ to R⁸directly or as hydrogen of the C₁₋₄ alkyl or further groups and ringsgiven by the definition of R and R¹ to R⁸ are replaced by L²-Z.

Furthermore, S⁰ may be optionally further substituted. In general, anysubstituent may be used as far as the cleavage principle is notaffected.

Preferably, one or more further optional substituents are independentlyselected from the group consisting of halogen; CN; COOR⁹; OR⁹; C(O)R⁹;C(O)N(R⁹R^(9a)); S(O)₂N(R⁹R^(9a)); S(O)N(R⁹R^(9a)); S(O)₂R⁹; S(O)R⁹;N(R⁹)S(O)₂N(R^(9a)R^(9b)); SR⁹; N(R⁹R^(9a)); NO₂; OC(O)R⁹;N(R⁹)C(O)R^(9a); N(R⁹)S(O)₂R^(9a); N(R⁹)S(O)R^(9a); N(R⁹)C(O)OR^(9a);N(R⁹)C(O)N(R^(9a)R^(9b)); OC(O)N(R⁹R^(9a)); T; C₁₋₅₀ alkyl; C₂₋₅₀alkenyl; or C₂₋₅₀ alkynyl, wherein T; C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; andC₂₋₅₀ alkynyl are optionally substituted with one or more R¹⁰, which arethe same or different and wherein C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀alkynyl are optionally interrupted by one or more groups selected fromthe group consisting of T, —C(O)O—; —O—; —C(O)—; —C(O)N(R¹¹)—;—S(O)₂N(R¹¹)—; —S(O)N(R¹¹)—; —S(O)₂—; —S(O)—; —N(R¹¹)S(O)₂N(R^(11a))—;—S—; —N(R¹¹)—; —OC(O)R¹¹; —N(R¹¹)C(O)—; —N(R¹¹)S(O)₂—; —N(R¹¹)S(O)—;—N(R¹¹)C(O)O—; —N(R¹¹)C(O)N(R^(11a))—; and —OC(O)N(R¹¹R^(11a));

R⁹, R^(9a), R^(9b) are independently selected from the group consistingof H; T; and C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; or C₂₋₅₀ alkynyl, wherein T;C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionally substitutedwith one or more R¹⁰, which are the same or different and wherein C₁₋₅₀alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionally interrupted byone or more groups selected from the group consisting of T, —C(O)O—;—O—; —C(O)—; —C(O)N(R¹¹)—; —S(O)₂N(R¹¹)—; —S(O)N(R¹¹)—; —S(O)₂—; —S(O)—;—N(R¹¹)S(O)₂N(R^(11a))—; —S—; —N(R¹¹)—; —OC(O)R¹¹; —N(R¹¹)C(O)—;—N(R¹¹)S(O)₂—; —N(R¹¹)S(O)—; —N(R¹¹)C(O)O—; —N(R¹¹)C(O)N(R^(11a))—; and—OC(O)N(R¹¹R^(11a));

T is selected from the group consisting of phenyl; naphthyl; indenyl;indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; 4 to 7 membered heterocyclyl; or9 to 11 membered heterobicyclyl, wherein T is optionally substitutedwith one or more R¹⁰, which are the same or different;

R¹⁰ is halogen; CN; oxo (═O); COOR¹²; OR¹²; C(O)R¹²; C(O)N(R¹²R^(12a));S(O)₂N(R¹²R^(12a)); S(O)N(R¹²R^(12a)); S(O)₂R¹²; S(O)R¹²;N(R¹²)S(O)₂N(R^(12a)R^(12b)); SR¹²; N(R¹²R^(12a)); NO₂; OC(O)R¹²;N(R¹²)C(O)R^(12a); N(R¹²)S(O)₂R^(12a); N(R¹²)S(O)R^(12a);N(R¹²)C(O)OR^(12a); N(R¹²)C(O)N(R^(12a)R^(12b)); OC(O)N(R¹²R^(12a)); orC₁₋₆ alkyl, wherein C₁₋₆ alkyl is optionally substituted with one ormore halogen, which are the same or different;

R¹¹, R^(11a), R¹², R^(12a), R^(12b) are independently selected from thegroup consisting of H; or C₁₋₆ alkyl, wherein C₁₋₆ alkyl is optionallysubstituted with one or more halogen, which are the same or different.

The term “interrupted” means that between two carbons a group isinserted or at the end of the carbon chain between the carbon andhydrogen.

L² is a single chemical bond or a spacer. In case L² is a spacer, it ispreferably defined as the one or more optional substituents definedabove, provided that L² is substituted with Z.

Accordingly, when L² is other than a single chemical bond, L²-Z isCOOR⁹; OR⁹; C(O)R⁹; C(O)N(R⁹R^(9a)); S(O)₂N(R⁹R^(9a)); S(O)N(R⁹R^(9a));S(O)₂R⁹; S(O)R⁹; N(R⁹)S(O)₂N(R^(9a)R^(9b)); SR⁹; N(R⁹R^(9a)); OC(O)R⁹;N(R⁹)C(O)R^(9a); N(R⁹)S(O)₂R^(9a); N(R⁹)S(O)R^(9a); N(R⁹)C(O)OR^(9a);N(R⁹)C(O)N(R^(9a)R^(9b)); OC(O)N(R⁹R^(9a)); T; C₁₋₅₀ alkyl; C₂₋₅₀alkenyl; or C₂₋₅₀ alkynyl, wherein T; C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; andC₂₋₅₀ alkynyl are optionally substituted with one or more R¹⁰, which arethe same or different and wherein C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀alkynyl are optionally interrupted by one or more groups selected fromthe group consisting of -T-, —C(O)O—; —O—; —C(O)—; —C(O)N(R¹¹)—;—S(O)₂N(R¹¹)—; —S(O)N(R¹¹)—; —S(O)₂—; —S(O)—; —N(R¹¹)S(O)₂N(R^(11a))—;—S—; —N(R¹¹)—; —OC(O)R¹¹; —N(R¹¹)C(O)—; —N(R¹¹)S(O)₂—; —N(R¹¹)S(O)—;—N(R¹¹)C(O)O—; —N(R¹¹)C(O)N(R^(11a))—; and —OC(O)N(R¹¹R^(11a));

R⁹, R^(9a), R^(9b) are independently selected from the group consistingof H; Z; T; and C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; or C₂₋₅₀ alkynyl, wherein T;C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionally substitutedwith one or more R¹⁰, which are the same or different and wherein C₁₋₅₀alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionally interrupted byone or more groups selected from the group consisting of T, —C(O)O—;—O—; —C(O)—; —C(O)N(R¹¹)—; —S(O)₂N(R¹¹)—; —S(O)N(R¹¹)—; —S(O)₂—; —S(O)—;—N(R¹¹)S(O)₂N(R^(11a))—; —S—; —N(R¹¹)—; —OC(O)R¹¹; —N(R¹¹)C(O)—;—N(R¹¹)S(O)₂—; —N(R¹¹)S(O)—; —N(R¹¹)C(O)O—; —N(R¹¹)C(O)N(R^(11a))—; and—OC(O)N(R¹¹R^(11a));

T is selected from the group consisting of phenyl; naphthyl; indenyl;indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; 4 to 7 membered heterocyclyl; or9 to 11 membered heterobicyclyl, wherein t is optionally substitutedwith one or more R¹⁰, which are the same or different;

R¹⁰ is Z; halogen; CN; oxo (═O); COOR¹²; OR¹²; C(O)R¹²;C(O)N(R¹²R^(12a)); S(O)₂N(R¹²R^(12a)); S(O)N(R¹²R^(12a)); S(O)₂R¹²;S(O)R¹²; N(R¹²)S(O)₂N(R^(12a)R^(12b)); SR¹²; N(R¹²R^(12a)); NO₂;OC(O)R¹²; N(R¹²)C(O)R^(12a); N(R¹²)S(O)₂R^(12a); N(R¹²)S(O)R^(12a);N(R¹²)C(O)OR^(12a); N(R¹²)C(O)N(R^(12a)R^(12b)); OC(O)N(R¹²R^(12a)); orC₁₋₆ alkyl, wherein C₁₋₆ alkyl is optionally substituted with one ormore halogen, which are the same or different;

R¹¹, R^(11a), R¹², R^(12a), R^(12b) are independently selected from thegroup consisting of H; Z; or C₁₋₆ alkyl, wherein C₁₋₆ alkyl isoptionally substituted with one or more halogen, which are the same ordifferent;

provided that one of R⁹, R^(9a), R^(9b), R¹⁰, R¹¹, R^(11a), R¹²,R^(12a), R^(12b) is Z.

Even more preferred general aromatic structures are listed below.

wherein

NH-rhGH represents the rhGH residue attached to the transient linker;

R1, R2, R3, R4, and R5 are selected independently from hydrogen, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl,

PEG represents the PEGylation residue attached to the transient linker,

and n=1 or 2, and

X is selected from C1 to C8 alkyl or C1 to C12 heteroalkyl.

The term “C1 to C12 heteroalkyl” means an alkyl chain having 1 to 12carbon atoms which are optionally interrupted by heteroatoms, functionalgroups, carbocycles or heterocycles as defined above.

In a preferred embodiment, in formula (A) L^(a) is represented by thecarbamate group attached to rhGH, G^(a) is represented by the aromaticoxygen group, the carbonyl attached to it, and the substituent attachedto the carbonyl as shown in formula I.

More preferred structures are given by general formula I, which are partof the structure (A) within the general aromatic linker structure above:

and where preferred examples of formula I comprise:

More preferred aromatic structures of formula II, which are part of thestructure (A) within the general aromatic linker structure above:

and where preferred examples of formula II comprise:

More preferred structures of formula III, which are part of thestructure (A) within the general aromatic linker structure above,wherein PEG-X is

and PEG-W includes the following substituent groups:

One example of preferred prodrug conjugates are shown below:

R is selected from hydrogen, methyl, ethyl, propyl and butyl,

X is selected from C1 to C8 alkyl or C1 to C12 heteroalkyl.

Also in the preferred and more preferred embodiments PEG meanspreferably the rest of S⁰, comprising at least S¹, S², BS¹ andoptionally BS².

In a preferred embodiment prodrugs of the present invention are selectedfrom the group consisting of

wherein m is an integer from 200 to 250 and n is an integer from 100to125;

wherein n is an integer from 400 to 500;

wherein n is an integer from 400 to 500; and

wherein n is an integer from 400 to 500.

Prodrugs of the present invention can be prepared by methods known inthe art. However especially for compounds of formula (AA1) it ispreferred to build up the prodrug molecule in a convergent synthesis byproviding a first precursor molecule comprising one or more thiol groupsand an activated carbonate group and a second precursor moleculecomprising a maleimide group to react in an addition reaction resultingin the formation of a thio succinimide group and to react that combinedprecursor molecule with hGH to yield a compound of formula (AA1).

Accordingly, another aspect of the present invention is a method for thepreparation of a compound of formula hGH-NH—C(O)O—S⁰ (AA1), wherein S⁰has the meaning as indicated above and comprises at least one group

the method comprises the following steps:

-   -   (a) reacting a compound of formula ROC(O)O—S^(0′)—SH (AA1′) with        a compound of formula

wherein R is a suitable rest for an activated carbonate group andwherein S^(0′) and S^(0″) are selected to yield S⁰ comprising the atleast one group

resulting in a compound of formula ROC(O)O—S⁰, and

-   -   (b) reacting the compound of formula ROC(O)O—S⁰ with hGH-NH₂,        wherein hGH-NH₂ represents hGH with one of its primary amino        groups to yield a compound of formula (AA1).

Suitable R groups for the carbonate functional groups includesubstituted alkyl or carbocyclic or heterocyclic, like aryl orcycloalkyl, groups like the pentafluorophenyl or NHS group.

Assays to Determine the Functional Properties of rhGH PEGylated Prodrug

Activity and Half Life of the Conjugate

To determine the activity and the half life of the prodrug conjugatedescribed herein, it is necessary to synthesize a “permanent” conjugate,which does not undergo autohydrolysis—that is a permanent conjugate.

This is achieved by synthesizing a molecule identical to the rhGHPEGylated prodrug, apart from modifying the part of the linkerstructure, which initiates the autocleavage. Such corresponding compoundwill have residual activity and circulating half-life of the conjugateidentical to that of the rhGH PEGylated prodrug. More generally, for anyself hydrolysable (autocleavage) transient linker conjugated prodrugs itcan be envisioned to synthesize a molecule identical to the prodrug,apart for the ability to undergo autocleavage, by making minormodification to the linker structure.

The reason for using the corresponding conjugate is that if the selfhydrolysable (autocleavage) transient linker as described herein isapplied in the assay mentioned in Example 1, the conjugate willobviously immediately begin to release unmodified drug, which willinfluence assay results. In other words, it is not possible to measureresidual activity without preparing a permanent conjugate as theunchanged native drug e.g. rhGH, will be released and contribute to themeasured activity. This is obvious to the skilled person.

For this reason as explained above the residual activity of the selfhydrolysable (autocleavage) transient linker conjugated prodrugs areexpressed as the activity of the corresponding permanent conjugates. Thepermanent conjugates are prepared in a similar fashion to the selfhydrolysable (autocleavage) transient linker conjugated prodrugs(examples 20 through 23), but with a minor modification in the linkerstructure, so that the linker no longer can undergo autocleavage. Thepreparation of permanent conjugates is described in Example 10 throughExample 19.

According to one embodiment of the present invention rhGH PEGylatedprodrug as described herein is characterized by that:

-   -   (1): when PEG is linked to rhGH in the prodrug conjugate the        prodrug has an GH activity with is less than 5% of the native        growth hormone without PEG to avoid injection side lipoatrophy;        and    -   (2): PEG is linked to rhGH via a self hydrolysable        (autocleavage) transient linker, wherein the linker        autohydrolysis rate is such that the in vivo half-life is from        10 hours to 600 hours.

The assay to determine property (1) is described in detail in workingexample 1 herein. Based on these detailed instructions it is routinework for the skilled person to measure this residual activity of theprodrug.

In a preferred embodiment the residual activity of property (1) is lessthan 5%, more preferably less than 3%, even more preferably less than 1%and most preferably virtually inactive.

The assay to determine property (2) is described in detail in workingexample 2 herein. Based on these detailed instructions it is routinework for the skilled person to measure this autocleavage rate of thetransient linker of the prodrug.

In a preferred embodiment the autocleavage rate in vivo half-life issuch that the in vivo half-life is from 20 hours to 300 hours, morepreferably from 20 hours to 150 hours, even more preferably from 30hours to 150 hours, even more preferably from 30 hours to 100 hours,even more preferably from 40 hours to 100 hours even more preferablyfrom 50 to 75 hours and also even more preferably from 30 to 75 hours.

In Vivo and in Vitro Correlation

It is known from previous patent applications from the company AscendisPharma (Complex Biosystems Company) that there is good correlationbetween in vitro and in vivo linker cleavage rates. In vivo releasekinetics can be readily predicted from the in vitro experimental data.

Lipoatrophy

As described above lipoatrophy is lipolysis occurring in close proximityof the injection site. Therefore, measuring in vitro lipolysis of growthhormone and growth hormone conjugates can be used to estimate thelipoatrophy effect of the conjugates.

To determine the lipolytic effect of the prodrug conjugate describedherein, it is necessary to synthesize a “permanent” conjugate, whichdoes not undergo autohydrolysis—that is a permanent conjugate.

The assay to determine lipoatrophy is described in details in workingexample 3 herein. Based on these detailed instructions it is routinework for the skilled person to measure lipoatrophy.

In a preferred embodiment, the rhGH PEGylated prodrug conjugate, asdescribed herein, has a lipoatrophy effect that is comparable to humangrowth hormone, measured according to the assay to determine lipoatrophyof example 3 and other identical dosage regimen conditions.

GH Related Diseases

The term “a GH related” disease of second aspect simply herein relatesto diseases and conditions where a human could benefit from GH.

This includes, but is not limited to, growth hormone deficiency, adultonset growth hormone deficiency, Turner syndrome, Prader-Willi syndrome,short bowel syndrome, chronic renal insufficiency, small for gestationalage (SGA), AIDS wasting, anti-ageing, rheumatoid arthritis, idiopathicsmall stature, short stature homeobox gene and somatopause. Included isalso other short stature condition, which includes Noonan syndrome,skeletal dysplasia, Down syndrome, short stature associated withprolonged steroid use, Aarskog's syndrome, among others.

Also included are chronic renal disease, juvenile rheumatoid arthritis;cystic fibrosis, HIV-infection in children receiving HAART treatment(HIV/HALS children); short stature in children born with very low birthweight (VLBW) but SGA; skeletal dysplasia; hypochondroplasia;achondroplasia; idiopathic short stature (ISS); GHD in adults; fracturesin or of long bones, such as tibia, fibula, femur, humerus, radius,ulna, clavicula, matacarpea, matatarsea, and digit; fractures in or ofspongious bones, such as the scull, base of hand, and base of food;patients after tendon or ligament surgery in e.g. hand, knee, orshoulder; distraction oteogenesis; disorders resulting from hip ordiscus replacement, meniscus repair, spinal fusions or prosthesisfixation, such as in the knee, hip, shoulder, elbow, wrist or jaw;disorders resulting from fixing of osteosynthesis material, such asnails, screws and plates; non-union or mal-union of fractures; disordersresulting from osteatomia, e.g. from tibia or 1st toe; disordersresulting from graft implantation; articular cartilage degeneration inknee caused by trauma or arthritis; osteoporosis in patients with Turnersyndrome; osteoporosis in men; adult patients in chronic dialysis(APCD); malnutritional associated cardiovascular disease in APCD;reversal of cachexia in APCD; cancer in APCD; chronic abstractivepulmonal disease in APCD; HIV in APCD; elderly with APCD; chronic liverdisease in APCD, fatigue syndrome in APCD; Crohn's disease; impairedliver function; males with HIV infections; short bowel syndrome; centralobesity; HIV-associated lipodystrophy syndrome (HALS); male infertility;patients after major elective surgery, alcohol/drug detoxification orneurological trauma; aging; frail elderly; osteo-arthritis;traumatically damaged cartilage; erectile dysfunction; fibromyalgia;memory disorders; depression; traumatic brain injury; subarachnoidhaemorrhage; very low birth weight; metabolic syndrome; glucocorticoidmyopathy; and short stature due to glucucorticoid treatment in children.

FIGURES

In the figures the following is shown.

FIG. 1 shows SDS-PAGE analysis of purified permanent PEG-hGH conjugates,wherein Lane 1: HiMark™ Pre-stained High Molecular Weight ProteinStandard; lane 2: compound 23; lane 3: compound 23; lane 4: compound 25;lane 5: compound 26, lane 6: compound 33; lane 7: compound 32; lane 8:compound 28; lane 9: compound 28; lane 10: compound 28; lane 11:compound 30; lane 12: compound 34; lane 13: compound 34; lane 14:compound 27; lane 15: compound 29.

FIG. 2 shows size exclusion chromatogram of quenched reaction solutionof the synthesis of conjugate 28 and size exclusion chromatogram ofpurified 28

FIG. 3 shows cation exchange chromatography purification of conjugate 35and size exclusion chromatogram of purified 35

FIG. 4 shows cation exchange chromatography purification of conjugate 36and size exclusion chromatogram of purified 36

FIG. 5 shows cation exchange chromatography purification of conjugate 37and size exclusion chromatogram of purified 37

FIG. 6 shows size exclusion chromatograms of samples of conjugate 35incubated in buffer at pH 7.4 and 37° C. at various time points

FIG. 7 to 10 show preferred prodrug conjugates of the present inventionindicating S⁰, S¹, S², L^(a), G^(a), BS¹, BS², BS³ and the criticaldistance.

FIG. 11 shows pharmacodynamic response curves of conjugate 36.

In detail, FIGS. 7 and 8 shows exemplary structures 35 and 38 of type(AA1, AAA1), where the at least 5 kDa polymer chain of S⁰ comprisingG^(a) and BS¹ and BS² is marked as S⁰; the carbamate group resultingfrom L^(a) and the primary amino group of hGH is marked as L^(a); BS¹comprises the at least 4 kDa polymer chain marked as S¹, wherein S¹comprises BS³, which comprises the at least 4 kDa polymer chain markedas S³. BS² comprises the at least 4 kDa polymer chain marked as S². Thecritical distance is given by 18 atoms for FIGS. 7 and 4 atoms for FIG.8.

In FIGS. 9 and 10, exemplary structures of the type (AA2, AAA2) areshown, wherein the amide group resulting from L^(a) and the primaryamino group of hGH is marked as L^(a) and the residue attached to L^(a)is marked S⁰ comprising G^(a) and “PEG” representing the rest of S⁰comprising at least BS¹, S¹ and S² (all not shown).

EXAMPLES

Methods

Analytical and Preparative RP-HPLC

Analytical RP-HPLC/ESI-MS was performed on Waters equipment consistingof a 2695 sample manager, a 2487 Dual Absorbance Detector, and a ZQ 4000ESI instrument equipped with a 5 μm Reprosil Pur 300 Å ODS-3 columns(75×1.5 mm) (Dr. Maisch, Ammerbuch, Germany; flow rate: 350 μL/min,typical gradient: 10-90% acetonitrile in water, 0.05% TFA over 5 min).

For preparative RP-HPLC a Waters 600 controller and a 2487 DualAbsorbance Detector was used equipped with the following columns(Reprosil Pur 300 Å ODS-3)

A): 100×20 mm, 10 mL/min flow rate, typical gradient: 10-90%acetonitrile in water, 0.1% TFA over 11 min

or

B): 100×40 mm (10 μm particles), 40 mL/min flow rate, typical gradient:10-90% acetonitrile in water, 0.1% TFA over 11 min.

Cation Exchange Chromatography

The purification of conjugates by cation exchange chromatography wasperformed using an ÄKTA Explorer system (GE Healthcare) equipped with aMacrocap SP column. The respective conjugate in 20 mM sodium acetatebuffer pH 4 was applied to the column that was pre-equilibrated in 20 mMsodium acetate buffer pH4 (buffer A). The column was washed with threecolumn volumes of Buffer A to remove any unreacted PEG reagent.Conjugates were eluted using a gradient of 10-60% buffer B (20 mM sodiumacetate, 1 M sodium chloride, pH 4.5) over 20 column volumes or 0-40%buffer B over 20 column volumes and then 40-80% B over three columnvolumes. The flow rate was 7 ml/min and the eluent was monitored bydetection at 280 nm.

Anion Exchange Chromatography

The purification of conjugates by anion exchange chromatography wasperformed using an ÄKTA Explorer system (GE Healthcare) equipped with aSource Q column. The respective conjugate in 20 mM Tris/HCl buffer pH7.5 (buffer C) was applied to the column that was pre-equilibrated inbuffer C. The column was washed with three column volumes of buffer C toremove any unreacted PEG reagent. Conjugates were eluted using agradient of 0-20% buffer D (20 mM Tris/HCl, 1 M sodium chloride, pH 7.5)over 25 column volumes. The flow rate was 5 ml/min and the eluent wasmonitored by UV detection at 280 nm. Alternatively, the buffer system 20mM bis-tris/HCl, pH 6.5 (buffer E) and 20 mM bis-tris/HCl, 1 M sodiumchloride, pH 6.5 (buffer F) was used.

Analytical Size Exclusion Chromatography

Analytical size exclusion chromatography analysis was performed on aÄKTA Explorer (GE Healthcare) system. Samples were analyzed using aSuperdex 200 or a Sepharose 6 column (10×300 mm) and 20 mM sodiumphosphate, 135 mM sodium chloride, pH 7.4 was used as mobile phase. Theflow rate for the column was 0.75 ml/min and the eluted hGH andpolymer-hGH conjugates were detected at 215 nm and 280 nm.

Activity Determination of pfp-Activated mPEG-Linker Reagents

A defined amount of pfp-activated mPEG-linker reagent (3-5 mg) wasdissolved in 100 μL WATER. 10 μL 0.5 M NaOH were added and the reactionmixture was reacted for 60 min at 40° C. 1.5 μL TFA were added and 10%of this mixture were analyzed by analytical RP-HPLC. The chromatogramswere recorded at 260 and 280 nm. The peak corresponding topentafluorophenol was integrated. Determined values were compared withan appropriate calibration curve generated by analyzing defined amountsof pfp by analytical RP-HPLC and integration of chromatograms recordedat 260 and 280 nm.

SDS-PAGE Analysis

The permanent mPEG-hGH conjugates were analysed using NuPAGE® NovexTris-Acetate gels (1.5 mm thick, 15 lanes), NuPAGE Tris-AcetateSDS-Running Buffer, HiMark™ Pre-stained High Molecular Weight ProteinStandard and Simply Blue™ SafeStain (Invitrogen). In each lane 0.2-0.6μg conjugate were applied and the electrophoresis and subsequentstaining performed according to the supplier's protocol.

Example 1 Assay to Measure hGH PEGylated Prodrug and hGH Activity

The biological activity of hGH is measured by using standard assaysknown to the skilled person in the art. As described in EP1715887B1 andas also discussed above, the biological activity associated with thenative or modified hGH (for example a PEGylated hGH), can be measuredusing standard FDC-P1 cell proliferation assays, (Clark et al, Journalof Biological Chemistry 271: 21969-21977) or receptor binding assay(U.S. Pat. No. 5,057,417).

On line 8 (page 14) of patent EP1715887B1, it is described that thepreferred in vitro activity has to be as high as possible, mostpreferred the modified hGH has equivalent or improved in vitrobiological activity. In current invention, the biological activity hasto be as low as possible compared to native hGH. Thus current inventorsdid the complete opposite compared to the prior art described inEP1715887B1.

In Vitro Assay

The in vitro activities of the permanent PEG-hGH conjugates described inthe examples below are determined using one or more standard assays forassessing biological activity in vitro. Standard assays that may beemployed include cell proliferation assays using, e.g., FDC-PI cells(see, e.g., Clark et al., Journal of Biological Chemistry,271:21969-21977, 1996), or Ba/F3-hGHR cells, which express receptors forhGH, hGH delta 135-146, or Nb2 rat lymphoma cells, which proliferate inresponse to hGH via the lactogenic receptors (see, e.g., Alam, K. S., etal., J. Biotech 2000, Feb. 28, 78(1), 49-59). Receptor binding assays(see, e.g., U.S. Pat. No. 5,057,417) may also be used.

Nb2-11 is a clone of the Nb-2 rat lymphoma line which was derived from atransplant of a lymphoma that developed in the thymus/lymph node of amale noble (Nb) strain rat following prolonged oestrogen treatment. Thecells are of the pre-T cell origin and their proliferation is dependenton mammalian lactogens, such as prolactin. Nb2-11 can also bemitogenically stimulated by IL-2. Injection of Nb2 cells into Nb ratsgives rise to malignant tumors that are highly sensitive to treatmentwith vinca alkaloids. Karyotypic analysis has shown that the cell linehas only five well developed chromosome abnormalities. The cells do notexpress surface immunoglobulin, and their lactogens dependency isconfirmed. Protocols for the use of Nb2-11 cells in bioassays areavailable from ECACC on request.

As WO2006102659 describes on page 74 paragraph 0240 example 7, thebiological activity of hGH and the conjugates described herein shall beassessed in vitro using an NB2-11 rat lymphoma cell proliferation assay.Briefly, NB2-11 cells derived from a rat lymphoma are incubated withhGH, which lead to binding of the hGH molecule to its receptor on thecell surface. Receptor binding induces the signal transduction cascade,which results in proliferation of the cells. Assay results are based ondetermined protein content, and a 100% bioactivity of unmodified hGH.

Conclusion:

Based on detailed instructions of this example 1 it is routine work forthe skilled person to measure this residual activity of the prodrug.

Example 2 Assay to Measure Autocleavage Rate of the Transient Linker ofthe Prodrug

Determination of in Vitro Half-Life

For determination of in vitro linker cleavage rate of PEG-linker-hGHconjugates, the compounds are dissolved in buffer at pH 7.4 (e.g. 10 mMsodium phosphate, 140 mM NaCl, 3 mM EDTA) and solution is filteredthrough a 0.22 μm filter and incubated at 37° C. Samples are taken attime intervals and analyzed by RP-HPLC or size exclusion chromatographyat 215 nm. Peaks corresponding to liberated hGH are integrated andplotted against incubation time. Curve fitting software is applied todetermine first-order cleavage rates.

In Vivo Half-Life Determination and in Vitro/in Vivo Half-LifeCorrelation

Linker cleavage rates in vivo are determined by comparing thepharmacokinetics of permanent PEG-hGH conjugates with the respectivetransient PEG-linker-hGH conjugate carrying the same PEG moiety afterintravenous injection into rat.

Firstly, permanent PEG-hGH conjugate is injected intravenously into ratsand blood samples are taken at time intervals, plasma prepared, andanalyzed for hGH using an ELISA.

Secondly, transient PEG-hGH conjugate is injected intravenously in rats,blood samples are taken at time intervals, plasma prepared, and analyzedfor hGH using an ELISA.

In vivo half-life is calculated from the ratio of hGH concentration oftransient conjugate divided by determined hGH concentration of permanentconjugate at the respective time points and curve fitting. Data arecompared to in vitro half-life measurements.

Conclusion

Based on detailed instructions of this example 2 it is routine work forthe skilled person to measure the in vivo half-life of the hGH-PEGylatedprodrug.

Example 3 Assay to Measure Lipoatrophy

As said above compound PHA-794428 is a PEGylated-hGH and described inpatent EP1715887 from the company Pharmacia. According towww.clinicaltrials.gov, the study was terminated on 10 Dec. 2007.Pfizer's (Pharmacia) decision to terminate the program was due to casesof injection-site lipoatrophy that were reported in the clinical Phase 2studies after a single injection of PHA 794428. Lipoatrophy is the termdescribing the localized loss of fat tissue and is visible on humans asholes in the skin (visible by the eye).

Assay

There are several in vitro methods described in the art in measurelipoatrophy. One proposal is described in publication J. Anim. Sci(1972), 35: 794-800 (L. J. Machlin) on page 795. Another description isfound in Int. J. Cancer; 80, 444-447 (1999).

Generally, lipoatrophy can be measured as proposed below.

Lipolytic effect can be determined using an in vitro assay consisting ofisolated mammal adipocytes, preferable murine adipocytes. Samples to beassayed were incubated at physiologically relevant conditions with apredetermined number of adipocytes in Krebs-Ringer bicarbonate buffercontaining appropriate nutrients for up to 6 hours. The concentration ofreleased glycerol is determined by standard methods, for exampleenzymatically or by a radiometric detection. Control samples containingadipocytes alone are analyzed to determine the spontaneous glycerolrelease.

The lipolytic effect of native unmodified recombinant human growthhormone and permanently PEGylated recombinant human growth hormone iscompared to that of transiently PEGylated recombinant human growthhormone.

Conclusion

Based on detailed instructions of this example 3 it is routine work forthe skilled person to measure the lipoatrophy effect.

Example 4 Synthesis of Permanent Linker Reagent 12a and Transient LinkerReagents 12b and 12c

Synthesis of Compound 6

6-Amino-hexan-1-ol (2.85 g, 24.3 mmol) was dissolved in aq. HBr (48%, 10mL, 89 mmol) and stirred at 80° C. for 3 h. Excess HBr was evaporated at50-65° C. and 15 Torr and the residue was dried in vacuo.

1: Yield 6.07 g (96%)

MS [M+H]⁺=180.3 g/mol (MW+H calculated=180.0 g/mol)

DBU (3.5 mL, 23.2 mmol) was added to a suspension of 6-Bromohexylaminehydrobromide 1 (3.03 g, 11.6 mmol) and triphenyl-methanethiol (2.14 g,7.74 mmol) in DCM (25 mL) and DMSO (13 mL) were added. The reactionmixture was stirred for 30 min at room temperature and diluted withwater (150 mL).

The aqueous layer was extracted with ether and the combined organicphase was evaporated. 2 was purified by RP-HPLC.

2: Yield 1.17 g (40%)

MS [M+H]⁺=376.7 g/mol (MW+H calculated=376.2 g/mol)

DBU (4.56 mL, 30.0 mmol) was added to 6-bromo-hexanoic acid (3.90 g,20.0 mmol) and triphenyl-methanethiol (11.1 g, 40.0 mmol) in DCM (40mL). After stirring at room temperature for 1 h ice cold 1 M H₂SO₄ (50mL) was added and the mixture was extracted with DCM. The combinedorganic phase was dried over Na₂SO₄ and concentrated in vacuo. Compound3 was purified by silica gel column chromatography (200 mL) usingheptane/ethyl acetate (4/1, R_(f)=0.2) as mobile phase.

3: Yield 5.83 g (75%)

DMAP (37 mg, 0.31 mmol) was added to 6-tritylsulfanyl-hexanoic acid 3(5.83 g, 14.9 mmol), thiazolidine-2-one (3.56 g, 29.9 mmol), anddicyclohexylcarbodiimide (3.08, 14.9 mmol) in DCM (100 mL). Afterstirring at room temperature for 1 h 1 M HCl (0.6 mL) was added and themixture was filtered. The filtrate was concentrated in vacuo and 4 waspurified by silica gel column chromatography (180 mL) usingheptane/ethyl acetate (1/1) as mobile phase.

4: Yield 7.15 g (97%) as yellow oil

A solution of 1-(2-thioxo-thiazolidin-3-yl)-6-tritylsulfanyl-hexan-1-one4 (1.53 g, 3.11 mmol) in THF (13 mL) was added over a period of 2 min to6-tritylsulfanyl-hexylamine 2 (1.17 g, 3.11 mmol) in DMSO (1 mL) and THF(5 mL). After addition of triethylamine (435 μL, 3.11 mmol) the reactionmixture was stirred for 90 min at room temperature. Ether (200 mL) andwater (100 mL) were added and the phases separated. After extraction ofthe aqueous phase with ether the combined organic phases were dried overNa₂SO₄ and concentrated in vacuo. Compound 5 was purified by silica gelcolumn chromatography (150 mL) using heptane/ethyl acetate (2/1,R_(f)=0.1) as mobile phase.

5: Yield 1.23 g (53%)

MS [M+Na]⁺=770.6 g/mol (MW+Na calculated=770.4 g/mol)

Under nitrogen, a 1M solution of LiAlH₄ in THF (1.2 ml, 4.8 mmol) wasplaced in a dry flask, and a solution of 5 (509 mg, 0.680 mmol) in 10 mlof THF was added over 4 min. The mixture was stirred under reflux for 2h, until complete conversion of the starting material was shown by thinlayer chromatography (heptanes/ethyl acetate 1:1). The reaction mixturewas carefully quenched with a 10:1 suspension of water in diethyl etheruntil the gas evolution had stopped. The mixture was poured into 50 mlof a saturated solution of sodium potassium tartrate and stirred for 90min. 90 ml of ethyl acetate were added and the phases were separated.The aqueous phase was extracted with ethyl acetate (4×20 ml), and thecombined organic phase was washed with brine (30 ml), dried over MgSO₄,filtered, and concentrated to give a transparent oil. 6 was adsorbed onsilica and purified by flash chromatography (30 g silica, CH₂Cl₂/MeOH20:1 (v/v)+0.1% NEt₃). The product was obtained as an off-white viscousoil.

6: Yield 270 mg (54%)

MS [M+H]⁺=734.4 g/mol (MW+H calculated=734.4 g/mol)

R_(f)=0.28 (CH₂Cl₂/MeOH 19:1)

Synthesis of Compound 9

AlCl₃ (23.0 g, 172.3 mmol) was added to glutaric anhydride (10.0 g, 86.2mmol) in anisole (85 mL, 781 mmol). The reaction mixture was heated to110° C. for 2 h, cooled to room temperature, poured on 3 N HCl/ice andextracted with dichloromethane. The aqueous phase was extracted withdichloromethane (4×20 ml), and the combined organic fractions werewashed with brine (30 ml), dried over MgSO₄, filtered and concentratedto give a red oil that was recrystallized from toluene. Product 7 wasobtained as an off-white solid.

7: Yield 5.2 g (48%)

MS [M+Na]⁺=245.8 (MW+Na calculated=245.1 g/mol)

AlCl₃ (9.0 mg, 68 mmol) was added to 7 (5.0 g, 23 mmol) in1,2-dichloroethane. The reaction mixture was stirred for 14 h at 85° C.and subsequently cooled to room temperature. Ice cold 1 N HCl (50 mL)was added and the mixture was extracted with ethyl acetate (4×30 mL).The combined organic fractions were dried over Na₂SO₄, filtered andconcentrated in vacuo to give a light red solid that was used in thenext step without further purification.

8: Yield 3 g (62%)

MS [M+H]⁺=209.1 (MW+H calculated=209.1 g/mol)

dicyclohexylcarbodiimide (532 mg, 2.6 mmol), acid 8 (403 mg, 1.9 mmol),HOSu (297 mg, 2.6 mmol), and collidine (1.0 mL, 7.8 mmol) in DCM (10 mL)were stirred for 90 min at room temperature. After removal ofdicyclohexylurea by filtration, amine 6 (947 mg, 1.3 mmol) in DCM (5 mL)and DIEA (450 μL, 2.6 mmol) were added to the filtrate and the mixturewas reacted for 14 h at room temperature. 1 N H₂SO₄ (2×50 mL) was addedand the phases were separated. The aqueous phase was extracted withethyl acetate (4×20 ml), and the combined organic phase was washed withbrine (30 ml), dried over MgSO₄, filtered and concentrated in vacuo. Theresidues were purified by silica gel column chromatography (150 mL)using heptane/ethyl acetate (1/2, R_(f)=0.66) as mobile phase.

9: Yield 819 mg (69%)

MS [M+Na]⁺=946.4 (MW+Na calculated=946.4 g/mol)

Synthesis of Permanent Linker Reagent 12a and Transient Linker Reagent12b and 12c

9 (1 eq., 175 mg, 0.19 mmol) was dissolved in dry THF (1.5 mL),p-nitrophenylchloroformate (1.1 eq., 42 mg, 0.21 mmol) and DIPEA (2 eq.,66 μl, 0.38 mmol) were added and the mixture was stirred for 60 min atroom temperature. Diethylamine (R1=R2=Et, 2 eq., 39 μl, 0.38 mmol) wasadded and stirring was continued for 15 min. The solvent was removed invacuo, 100 μl of AcOH were added and 10a was purified by RP-HPLC.

MS [M+Na]⁺=1045.9 (MW+Na calculated=1045.5 g/mol)

NaBH₄ (5 eq., 37 mg, 0.95 mmol) was added to 10a containing HPLCfraction (acetonitrile/H20˜3/1 (v/v)+0.1% TFA) and the mixture wasreacted for 10 min at room temperature. An additional portion of NaBH₄(5 eq., 37 mg, 0.95 mmol) was added and the reaction mixture was stirreduntil complete conversion of the starting material was indicated byLC/MS analysis (10 min at room temperature). 11a was purified by RP-HPLCand lyophilized.

11a: Yield 95 mg (49% based on 9)

MS [M+Na+H]⁺=1047.7 (MW+Na calculated=1047.5 g/mol)

9 (1 eq., 175 mg, 0.19 mmol) was dissolved in dry THF (1.5 mL),p-nitrophenylchloroformate (1.1 eq., 42 mg, 0.21 mmol) and DIPEA (2 eq.,66 μl, 0.38 mmol) were added and the mixture was stirred for 60 min atroom temperature. N,N,N′-Triethyl-ethane-1,2-diamine (R1=Et,R2=2-(diethylamino)ethyl, 2 eq., 68 μl, 0.38 mmol) was added andstirring was continued for 15 min. 100 μl of AcOH were added, thesolvent was removed in vacuo and 10b was purified by RP-HPLC andlyophilized.

10b: Yield 147 mg as TFA salt (65%)

MS [M+Na]⁺=1116.4 (MW+Na calculated=1116.6 g/mol)

10c was synthesized as described above usingN,N,N′-trimethyl-propane-1,3-diamine (R1=Me, R2=3-(dimethylamino)propyl,56 μL, 0.38 mmol) as diamine.

10c: Yield 134 mg as TFA salt (59%)

MS [M+Na]⁺=1088.4 (MW+Na calculated=1088.6 g/mol)

NaBH₄ (46 mg, 1.2 mmol) was added to 10b (147 mg, 0.12 mmol) inMeOH/water=95:5 (v/v) (3 mL) in two doses and the mixture was stirredfor 1 h at room temperature. After addition of AcOH (300 μL) andconcentration, product 11b was purified by RP-HPLC and lyophilized.

11b: Yield 107 mg as TFA salt (73%)

MS [M+Na]⁺=1118.4 (MW+Na calculated=1118.6 g/mol)

11c was synthesized according to the same protocol.

11c: Yield 65 mg as HCl salt (54%) from 134 mg starting material

MS [M+Na]⁺=1090.4 (MW+Na calculated=1090.6 g/mol)

Under a nitrogen atmosphere bis-pentafluorophenyl-carbonate (2.5 eq., 25mg, 63 μmol), DMAP (1 mg), and DIEA (5 eq., 22 μL, 127 μmol) were addedto 11a (1 eq., 26 mg, 26 μmol) in dry acetonitrile (0.5 mL). Thereaction mixture was stirred for 30 min at room temperature, cooled to0° C., and acidified with AcOH (200 μL). Product 12a was purified byRP-HPLC and lyophilized.

12a: Yield 13 mg (42%)

MS [M+Na]⁺=1258.2 (MW+Na calculated=1257.5 g/mol)

12b and 12c were prepared accordingly from 11b (56 mg, 48 μmol) and 11c(88 mg, 73 μmol), respectively.

12b: Yield 63 mg as TFA salt (93%)

MS [M+H]⁺=1306.3 (MW+H calculated=1306.6 g/mol)

12c: Yield 41 mg as TFA salt (41%)

MS [M+H]⁺=1278.4 (MW+Na calculated=1278.5 g/mol)

Example 5 Synthesis of Permanent Linker Reagent 14a and Transient LinkerReagents 14c

13a and13c were synthesized as described in WO2005/099768A2.

Under an atmosphere of nitrogen bispentafluorophenylcarbonate (631 mg,1.6 mmol), DMAP (20 mg, 0.16 mmol), and DIEA (556 μL, 3.2 mmol) wereadded to 13a (364 mg, 0.64 mmol) in dry acetonitrile (5 mL). Thereaction mixture was stirred for 15 min at room temperature, cooled to0° C., and acidified with acetic acid (1 mL). Product 14a was purifiedby RP-HPLC and lyophilized.

14a: Yield 379 mg (77%)

MS [M+Na]⁺=800.4 (MW+Na calculated=800.3 g/mol)

14c was prepared accordingly from 13c (97 mg, 130 μmol).

14c: Yield 114 mg as TFA salt (94%)

MS [M+H]⁺=821.5 (MW+H calculated=821.3 g/mol)

Example 6 Synthesis of Permanent Linker Reagent 15

Glutaric anhydride (0.41 mmol), amine 6 (200 mg, 0.27 mmol), DIPEA (72μL, 0.41 mmol), and DMAP (11 mg, 0.09 mmol) were stirred in acetonitrile(3 mL) for 2 h at 80° C. The mixture was cooled to room temperature andacetic acid (200 μL) was added. Product 15 was purified by RP-HPLC andlyophilized.

15: Yield 130 mg (57%)

MS [M+Na]⁺=870.2 (MW+Na calculated=870.4 g/mol)

Example 7 Synthesis of Activated mPEG-Linker Reagents

mPEG-maleimide starting materials:

mPEG residues after reacting with thiol group (R3 in structures below):

The vertical dashed line denotes the attachment site to the thiol groupin the respective structure

Synthesis of Permanent pfp-Activated mPEG-Linker Reagents 17aa, 17ab,17ac, 17ad, and Transient pfp-Activated mPEG-Linker Reagents 17b, 17ca,and 17cb

Carbonate 12a (13 mg, 10 μmol) was stirred in 10 μL AcOH, 700 μL HFIP, 1μL TFA and 2 μL TES for 10 min at room temperature. The volatilecomponents were removed in a nitrogen stream and 16a was purified byRP-HPLC.

16a: Yield 3.8 mg (5 μmol)

MS [M+H]⁺=751.3 (MW+H calculated=751.3 g/mol)

16b and 16c were prepared accordingly from 12b (7.7 mg, 5.4 μmol) and12c (2 mg, 1.5 μmol), respectively.

16b: Yield 2.5 mg (2.7 μmol)

MS [M+Na]⁺=845.1 (MW+Na calculated=844.3 g/mol)

16c: Yield 0.5 mg (0.6 μmol)

MS [M+Na]⁺=816.6 (MW+Na calculated=816.3 g/mol)

mPEG-maleimide 1B (NOF, Japan) (521 mg, 12.7 μmol) was added to 3.5 mg(3.9 μmol) 16c in 4 mL 3/1 (v/v) acetonitrile/water+0.1% TFA. 200 μL of0.5 M phosphate buffer pH 7.4 were added and the mixture was reacted for10 min at room temperature. 1 μL (13 μmol) mercaptoethanol were addedand the reaction mixture was acidified to pH 4-5 by addition of TFA. 17ca was purified by RP-HPLC and lyophilized.

17ca: Yield 220 mg (pfp-carbonate activity 82%)

17cb was synthesized as described for 17ca using 16c (3.5 mg, 3.9 μmol)and mPEG-maleimide 2B (656 mg, 16 μmol).

17cb: Yield 130 mg (pfp-carbonate activity 85%)

184 mg (8.8 μmol) mPEG-maleimide 1A (NOF, Japan) were added to 16a (2.0mg, 2.7 μmol) in 4 mL 1/1 (v/v) acetonitrile/water+0.1% TFA. 200 μL of0.5 M phosphate buffer pH7.4 were added and the mixture was reacted for10 min at room temperature. 0.2 μL (1.6 μmol) mercaptoethanol were addedand the reaction mixture was acidified to pH 2-3 by addition of TFA.17aa was separated from unreacted PEGs by RP-HPLC and lyophilized.

17aa: Yield 90 mg (pfp-carbonate activity 88%)

17ab was synthesized as described above using 16a (3.8 mg, 5.0 μmol) and680 mg (16 μmol) mPEG-maleimide 1B (NOF, Japan).

17ab: Yield 250 mg (pfp-carbonate activity 83%)

17ac was synthesized as described above using 16a (2.5 mg, 3.3 μmol) and200 mg (9.5 μmol) mPEG-maleimide 2A (Jenkem, PR China).

17ac: Yield 80 mg (pfp-carbonate activity 80%)

17ad can be synthesized as described above using 16a and mPEG-maleimide2B.

17b can be synthesized as described for 17cb using 16b andmPEG-maleimide 1B.

Example 8 Synthesis of pfp-Activated Permanent mPEG Linker Reagents 19aaand 19ab and Transient Permanent mPEG-Linker Reagent 19c

Carbonate 14c (20 mg, 21 μmol) was stirred in 10 μL AcOH, 400 μL HFIP,and 5 μL TES for 10 min at room temperature and cooled to 0° C. Ice coldacetonitrile/water=9/1 (v/v) was added and 18c was separated by RP-HPLCand lyophilized.

18c: Yield 5.0 mg as TFA salt (7.2 μmol)

MS [M+H]⁺=579.6 (MW+H calculated=579.2 g/mol)

18a was synthesized as described above using carbonate 14a (24 mg, 31μmol).

18a: Yield 8.0 mg (15 μmol)

MS [M+H]⁺=536.2 (MW+H calculated=536.5 g/mol)

205 mg (5 μmol) mPEG-maleimide 3A (NOF, Japan) were added to 18a (4.0mg, 7.5 μmol) in 2 mL 1/1 (v/v) acetonitrile/water+0.1% TFA. 100 μL of0.5 M phosphate buffer (pH7.4) were added and the mixture was reactedfor 10 min at room temperature. The reaction mixture was acidified to pH2-3 by addition of TFA and 19aa was separated from unreacted PEGs byRP-HPLC and lyophilized.

19aa: Yield 125 mg (pfp-carbonate activity 85%)

19ab was prepared accordingly from 410 mg (5 μmol) mPEG-maleimide 3B(NOF, Japan) and 18a (4.0 mg, 7.5 μmol).

19ab: Yield 265 mg (pfp-carbonate activity 87%)

19c was prepared accordingly from 205 mg mPEG-maleimide 3A and 18c (5mg, 7.2 μmol)

19c: Yield 120 mg (pfp-carbonate activity 88%)

Example 9 Synthesis of Permanent 4-Arm Branched 80 kDa mPEG-NHS EsterDerivative 22

Acid 15 (12 mg, 14 μmol) was stirred in 1 mL TFA, 1 mL DCM, and 10 μLTES for 10 min at room temperature. The volatile components were removedin a nitrogen stream and the dithiol 20 was purified by RP-HPLC.

20: Yield 2.9 mg (8 μmol)

MS [M+Na]⁺=386.8 (MW+Na calculated=386.2 g/mol)

20 (1 mg, 2.8 μmol) in 170 μL acetonitrile was added to mPEG-maleimide1B (NOF, Japan) (380 mg, 9.2 μmol) in 4 mL 1/1 (v/v)acetonitrile/water+0.1% TFA. 200 μL of 0.5 M phosphate buffer pH7.4 wereadded and the mixture was reacted for 10 min at room temperature. 0.6 μL(7.8 μmol) mercaptoethanol were added and the reaction mixture wasacidified to pH 4-5 by addition of TFA. The buffer was exchanged to0.005% HCl (HiPREP Desalting column, 26/10 GE healthcare) and 21 waslyophilized without further purification.

21: Yield 320 mg

21 was dissolved in 50 mL of toluene and the polymer solution wasazeotropically dried for two hours under reflux using a Dean-Stark trap.The polymer solution was then cooled to room temperature. The driedmPEG-linker reagent 21 was precipitated by addition of chilled ether (60mL).

dicyclohexylcarbodiimide (1.2 mg, 6 μmol) in DCM was added to a solutionof 21 (240 mg, 3 μmol) and N-hydroxy-succinimide (0.7 mg, 6 μmol) in DCM(3 mL). The reaction mixture was stirred for 14 h at room temperatureand 22 was precipitated by addition of cold ether (20 mL). Product 22was dried in vacuo.

22: Yield 200 mg

Example 10 Synthesis of Permanent Amide-Linked mPEG-hGH Monoconjugate 23and mPEG₂-hGH Bisconjugate 24 using Linear 40 kDa mPEG-SuccinimidylHexanoate Derivative

hGH was buffer exchanged to 50 mM sodium borate pH 8.5 (alternativelysodium borate pH 8 or sodium borate pH 9 can be used). The concentrationof hGH was approximately 2.5 mg/ml. A three-fold molar excess of 40 kDamPEG-succinimidyl hexanoate derivative (NOF, Japan) relative to theamount of hGH was dissolved in water to form a 20% (w/v) reagentsolution (alternatively a four-fold or five-fold molar excess can beused). The reagent solution was added to the hGH solution and mixed. Thereaction mixture was incubated for 2 h at room temperature and quenchedwith hydroxylamine at room temperature and pH 7 for two hours. Thequenched reaction mixture was analyzed by size exclusion chromatography.The monoconjugate 23 and bisconjugate 24 were purified by cationexchange chromatography. Alternatively, anion exchange chromatographycan be used for purification. The purified conjugates were analyzed bySDS-PAGE (FIG. 1).

Example 11 Synthesis of Permanent Amide-Linked mPEG-hGH Monoconjugate 25and mPEG-hGH Bisconjugate 26 using Branched 40 kDa mPEG-NHS EsterDerivative

Permanent mPEG-hGH monoconjugate 25 and bisconjugate 26 were synthesizedaccording to the procedure described in Example 10 using branched 40 kDamPEG-NHS ester derivative (NOF, Japan). The purified conjugates wereanalyzed by SDS-PAGE (FIG. 1).

Example 12 Synthesis of Permanent Amide-Linked mPEG-hGH Monoconjugate 27using 4-Arm Branched 80 kDa mPEG-NHS Ester Derivative

Permanent mPEG-hGH monoconjugate 27 was described according to Example10 using 4-arm branched 80 kDa mPEG-NHS ester derivative 22. Purified 27was analyzed by SDS-PAGE (FIG. 1).

Example 13 Synthesis of Permanent Carbamate-Linked mPEG-hGHMonoconjugate 28 using 4-Arm Branched 40 kDamPEG-Pentafluorophenylcarbonate Derivative 17aa

hGH was buffer exchanged to 50 mM sodium borate pH 9 (alternativelysodium borate pH 8.5 or sodium borate pH 8 can be used). Theconcentration of hGH was approximately 2.5 mg/ml. A four-fold molarexcess of permanent 4-arm branched 40 kDa mPEG-linker reagent 17aarelative to the amount of hGH was dissolved in water to form a 20% (w/v)reagent solution. The reagent solution was added to the hGH solution andmixed. The reaction mixture was incubated for 1.5 h at room temperatureand quenched by incubating in 100 mM hydroxylamine at pH 7 and roomtemperature for 2 h. The quenched reaction mixture was analyzed by sizeexclusion chromatography (FIG. 2 top). Permanent mPEG-linker-hGHmonoconjugate 28 was purified by anion exchange chromatography at pH 7.5and analyzed by SDS-PAGE (FIG. 1) and size exclusion chromatography(FIG. 2 bottom).

Example 14 Synthesis of Permanent Carbamate-Linked mPEG-hGHMonoconjugate 29 using 4-Arm Branched 80 kDamPEG-Pentafluorophenylcarbonate Derivative

Permanent carbamate-linked mPEG-hGH monoconjugate 29 was synthesizedaccording to Example 13 using 4-arm branched 80 kDamPEG-pentafluorophenyl carbonate derivative 17ab. Purified 29 wasanalyzed by SDS-PAGE (FIG. 1).

Example 15 Synthesis of Permanent Carbamate-Linked mPEG-hGHMonoconjugate 30 using 4-Arm Branched 40 kDamPEG-Pentafluorophenylcarbonate Derivative 17ac

Permanent mPEG-hGH monoconjugate 30 was synthesized according to Example13 using 4-arm branched 40 kDa mPEG-pentafluorophenyl carbonatederivative 17ac. Purified 30 was analyzed by SDS-PAGE (FIG. 1).

Example 16 Synthesis of Permanent mPEG-hGH Monoconjugate 31 using 4-ArmBranched 80 kDa mPEG-Pentafluorophenylcarbonate Derivative

Permanent mPEG-hGH monoconjugate 31 can be synthesized according toExample 13 using 4-arm branched 80 kDa mPEG-pentafluorophenyl carbonatederivative 17ad.

Example 17 Synthesis of Permanent Carbamate-Linked mPEG-hGHMonoconjugate 32 using 4-Arm Branched 40 kDamPEG-Pentafluorophenylcarbonate Derivative

Permanent carbamate-linked mPEG-hGH monoconjugate 32 was synthesizedaccording to Example 13 using 4-arm branched 40 kDamPEG-pentafluorophenyl carbonate derivative 19aa. Purified 32 wasanalyzed by SDS-PAGE (FIG. 1).

Example 18 Synthesis of Permanent Carbamate-Linked mPEG-hGHMonoconjugate 33 using 4-Arm Branched 80 kDamPEG-Pentafluorophenylcarbonate Derivative

Permanent mPEG-hGH monoconjugate 33 was synthesized according to Example13 using 4-arm branched 80 kDa mPEG-pentafluorophenyl carbonatederivative 19ab. Purified 33 was analyzed by SDS-PAGE (FIG. 1).

Example 19 Synthesis of Permanent Amine-Linked mPEG-hGH Monoconjugate 34using Branched 40 kDa mPEG-Propionaldehyde Derivative

hGH was buffer exchanged to 50 mM MES buffer pH 6 (alternatively HEPESbuffer pH 7 was used) and the concentration of hGH was adjusted to 1.5mg/ml. A three-fold molar excess of 40 kDa mPEG-propionaldehyde(GL2-400AL3, NOF, Japan) relative to the amount of hGH was dissolved inwater to form a 25% (w/v) reagent solution. The reagent solution wasadded to the hGH solution and mixed. An aliquot of a 1M stock solutionof sodium cyanoborohydride in water was added to give a finalconcentration of 25 mM in the reaction mixture. The solution wasincubated for 18 h at room temperature in the dark. The reaction wasquenched by the addition of Tris buffer. The reaction mixture wasanalyzed by size exclusion chromatography and conjugate 34 was purifiedby cation exchange chromatography. Purified mPEG-hGH monoconjugate 34was analyzed by SDS-PAGE (FIG. 1).

Example 20 Synthesis of Transient Carbamate-Linked mPEG-hGHMonoconjugate 35 using Transient 4-Arm Branched 40 kDamPEG-Pentafluorophenylcarbonate Derivative 19c

hGH was buffer exchanged to 50 mM sodium borate pH 9 (alternativelysodium borate pH 8.5 or sodium borate pH 8 can be used) and theconcentration of hGH was adjusted to 2.5 mg/ml. A four-fold molar excessof transient mPEG-linker reagent 19c relative to the amount of hGH wasdissolved in water to form a 20% (w/v) reagent solution. The reagentsolution was added to the hGH solution and mixed. The reaction mixturewas incubated for 1 h at room temperature and quenched by incubating in100 mM hydroxylamine at pH 7 and room temperature for 2 h.mPEG-linker-hGH monoconjugate was purified by anion exchangechromatography at pH 6.5 (FIG. 3 top) and analyzed by size exclusionchromatography (FIG. 3 bottom).

Example 21 Synthesis of Transient mPEG-Linker-hGH Monoconjugate 36 using4-Arm Branched 80 kDa mPEG-Pentafluorophenylcarbonate Derivative

hGH was buffer exchanged to 100 mM sodium borate pH 9 (alternativelysodium borate pH 8.5 or sodium borate pH 8 can be used) and theconcentration of hGH was adjusted to 10 mg/ml. A four-fold molar excessof transient 4-arm branched 80 kDa mPEG-linker reagent 17ca relative tothe amount of hGH was dissolved in water to form a 25% (w/v) reagentsolution. The reagent solution was added to the hGH solution and mixed.The reaction mixture was incubated for 45 min at room temperature andquenched by incubating in 100 mM hydroxylamine at pH 7 and roomtemperature for 2 h. mPEG-linker-hGH monoconjugate 36 was purified bycation exchange chromatography (FIG. 4 top) and analyzed by sizeexclusion chromatography (FIG. 4 bottom).

Example 22 Synthesis of Transient mPEG-hGH Monoconjugate 37 using 4-ArmBranched 80 kDa mPEG-Pentafluorophenylcarbonate Derivative 17cb

PEG-linker-hGH conjugate 37 was synthesized as described according tothe procedure described in Example 21 using activated mPEG-linkerreagent 17cb.

The cation exchange chromatogram and analytical size exclusionchromatogram are shown in FIG. 5 top and bottom, respectively.

Example 23 Synthesis of Transient Carbamate-Linked mPEG-hGHMonoconjugate 38 using 4-Arm Branched 80 kDamPEG-Pentafluorophenylcarbonate Derivative 17b

Transient carbamate-linked mPEG-linker-hGH conjugate 38 can besynthesized as described in Example 21 using transient 4-arm branched 80kDa mPEG-linker reagent 17b.

Example 24 Assay to Measure hGH PEGylated Prodrug and hGH Activity

It is routine work for the skilled person to determine the residualactivity of the polymeric prodrug as expressed by the activity of thecorresponding permanent polymer conjugate using standard assays asdescribed in example 1.

Specifically, NB2-11 cells were grown in serum free media with 100 ng/mlhGH supplement. For the in vitro proliferation assay, cell suspensioncontaining 2×10⁵ cells/ml were washed twice with serum free and hGH freemedium and dispensed into a 96-well flat bottom microtitre plate (10⁴cells/well). Compounds were tested in triplicate in a series oftitration steps (9 steps, using a factor 3 dilution between each step).The cells with compound solutions were incubated for 48 hours followedby incubation for 2.5 hours with cell proliferation reagent WST-1.NB2-11 proliferation was determined by optical density reading in anELISA reader and the response plotted as a function of concentration andEC50 values determined. The results are shown as % residual in vitrobioactivity in relation to unmodified hGH is provided in table 1.

In the in vitro experiments as described above, native hGH (source NovoNordisk, Denmark) was used as reference compound. The same hGHpreparation was used for the synthesis of the permanent PEG-hGHconjugates.

TABLE 1 Table 1: In vitro bioactivity of permanently PEGylated hGHconjugates as compared to native hGH (Norditropin, Novo Nordisk,Denmark). In vitro characterization: In vitro activity of permanentCompound conjugates Native hGH (hGH, Novo 100%  Nordisk, Denmark) 2310.3%  24 0.4% 25 4.4% 26 0.2% 27 0.7% 28 2.3% 29 0.7% 30 2.0% 32 6.3%33 2.2% 34 4.8%

Conclusion:

As seen from table 1, by conjugation of a suitable PEG molecule to hGH,the in vitro activity of the PEGylated hGH can be reduced to less than5% of the activity of the native unconjugated hGH. For example,conjugation of a branched PEG 4×20 kDa to hGH reduces the residualactivity to 0.7% of the unconjugated hGH standard.

Furthermore, from these results it was also surprisingly discovered,that the residual activity of the PEGylated growth hormone is relatednot only to the size of the attached PEG, but also to the degree ofbranching and the spacing between the hGH and the branching pointswithin the PEG structure.

Linear PEG

Specifically, attachment of a 40 kDa linear PEG to hGH results in an invitro activity of 10.3% (compound 23) compared to native hGH.

Branched PEG

When a branched 2×20 kDa PEG is attached (compound 25), the in vitroactivity is further reduced to 4.4% compared to native hGH.

Further, when a 4×20 kDa PEG with a short spacing between the hGH andthe branching points within the PEG reagent is attached (compound 27 and29) the in vitro activity is even further reduced to respectively 0.7%compared to native hGH.

Surprisingly, when a 4×20 kDa PEG with a relative long spacer betweenthe human growth hormone and the first branching point within the PEGreagent is attached (e.g. compound 33) the in vitro activity is lessreduced (2.2%) showing the importance of the spacer between the hGHfunctional group and the first branching point within the branched PEGreagent.

Conjugation of more than one PEG moiety to the hGH to formbisPEG-conjugates reduces the in vitro activity to lower than 0.5%.(e.g. compound 24 and 26).

Example 25 Determination of in Vitro Autocleavage Rate of Conjugate 35,36, 37, and 38

The autocleavage rate of conjugate 35, 36 and 37 at pH 7.4 and 37° C.was determined as described in Example 2. Autocleavage half-lives ofapproximately 75 h were determined for these conjugates. FIG. 6 showssize exclusion chromatograms of samples of incubated 35 analyzed after 0h, 8 h, 47 h, 97 h, and 168 h showing slow release of hGH from conjugate35 over time. Autocleavage rate of conjugate 38 can be determinedaccordingly and give half-lives of ca. 50 h.

Example 26 Assay to Measure Terminal in Vivo Half Life of the hGHPEGylated Prodrugs as Expressed by the Half Life of the CorrespondingPermanent Conjugate in Vivo

The pharmacokinetics of the permanent conjugates were determined afterintraveneous injection of 0.25 mg (hGH equivalents) into rats. In orderto select a conjugate suitable for weekly injections in humans, a plasmahalf life of more than 10 hours in the rat is desirable.

A single dose of 0.25 mg hGH or 0.25 mg permanent PEG-hGH conjugate(dose based on hGH) per rat was administered intravenously to maleWistar rats (200-250 g). Two groups of two animals each were used foreach compound. 200-300 μl whole blood was withdrawn sublingually toobtain 100 μl Ca-Heparin plasma per animal and time point. Samples werecollected after 0.5, 3, 24, 48, 72 and 96 h for group 1 and after 5, 8,32, 56, 80 and 168 h for group 2. Plasma samples were stored at −80° C.until assayed.

hGH and PEG-hGH conjugate concentrations were measured using a hGH ELISAkit (DSL). Plasma concentrations were calculated from a calibrationcurve of the respective conjugate or hGH and plotted against time, andthe terminal half-life (t_(1/2)) was calculated using a singlecompartment model. The result of the half life determination istabulated in table 2.

In order to select a conjugate suitable for weekly injections in humanspharmacokinetic studies in rats were performed. As the half life ofPEGylated conjugates in rats are in the range of 5 times faster than inhumans, the half life of a PEGylated hGH in rats should be about 10hours or longer. In order to obtain an estimate of the half life of theconjugated hGH PEGylated prodrug without linker cleavage, thepermanently conjugated corresponding conjugate is injected into rat.

The results of the in vivo half-life determinations are tabulated inTable 2.

TABLE 2 Table 2: Half life of permanent PEG-hGH conjugates in rats Invivo characterization: in vivo Compound half-life of permanentconjugates Native hGH (Novo 20 minutes Nordisk, Denmark) 23 4 hours 25 5hours 26 11 hours 27 13 hours

Conclusion:

From table 1 and table 2 it is obvious that residual activity correlatesinversely with half life e.g. a high degree of residual activity causesfaster elimination. This is typical for conjugates eliminated byreceptor mediated clearance mechanisms.

Furthermore, in order to obtain a hGH PEGylated prodrug that can beadministered once weekly in humans and with a low residual activity, aPEG molecule with one or more branching points and with a molecularweight of 40 kDa or above is preferred. Alternatively, conjugation ofPEG to more than one site on hGH to form bisPEG-hGH conjugates resultsin a long terminal half-life.

Example 27 Pharmacodynamic Study of Transient Carbamate-LinkedmPEG-Linker-hGH Conjugate 36 and Human Growth Hormone in CynomolgusMonkeys

The objective of this study was to compare the pharmacodynamic responsein cynomolgus monkeys of one dose of transient carbamate-linkedmPEG-linker-hGH conjugate 36 with once-daily human growth hormone dosingfor one week.

The following dosing groups were studied:

Dosing Test article Dose route Dose occasion Human growth hormone 0.3mg/kg/day SC Day 1, 2, 3, 4, 5, 6, 7 Transient carbamate-linked 5 mg/kgSC Day 1 mPEG-linker-hGH conjugate 36 Transient carbamate-linked 10mg/kg SC Day 1 mPEG-linker-hGH conjugate 36 Vehicle (10 mM succinic 0mg/kg SC Day 1 acid, 92 mg/mL trehalose, pH 4.0)

Since transient carbamate-linked mPEG-linker-hGH conjugate 36 istransiently PEGylated using a 80 kDa PEG group, the hGH amounts in the 5and 10 mg/kg transient carbamate-linked mPEG-linker-hGH conjugate 36dosing groups were approximately 1 and 2 mg/kg, respectively. Hence, thehGH amount in the 10 mg/kg group of transient carbamate-linkedmPEG-linker-hGH conjugate 36 was equivalent to a daily dose of 0.3 mg/kghGH.

Each test article was injected subcutaneously into 2 cynomolgus monkeys(1 male, 1 female) using a dose volume of 1 ml/kg. The age and weight ofthe animals were 2.5-3 years and 2.0-2.5 kgs, respectively.

Blood samples were collected from the femoral artery/vein fordetermination of serum concentrations of IGF-1, a pharmacodynamic markerfor human growth hormone. The blood sample were collected at thefollowing timepoints: 0 (predose), 3, 6, 12, 24, 36, 48, 72, 96, 120,and 144 hours after dosing on Day 1

Blood samples were collected, allowed to clot, and then stored on an iceblock or wet ice until centrifuged. After centrifugation, the serumsamples were aliquoted into prelabeled vials and tightly capped. Thevials were stored at −70° C. upon aliquoting into vials.

IGF-1 levels in the serum samples were measured using the QuantikineHuman IGF-1 ELISA kit (R&D systems) that had been adapted and validatedfor use in determining IGF-1 levels in cynomolgus monkey serum.

The pharmacodynamic response of the test articles is shown on FIG. 11.Both daily hGH administration and one administration of transientcarbamate-linked mPEG-linker-hGH conjugate 36 increased the IGF-1 levelsover the levels measured in the vehicle group. One administration of 5mg/kg transient carbamate-linked mPEG-linker-hGH conjugate 36 wasequivalent to daily hGH administration while one administration of 10mg/kg transient carbamate-linked mPEG-linker-hGH conjugate 36 was shownto be superior to daily hGH. This clearly indicated that a once-weeklydose of transient carbamate-linked mPEG-linker-hGH conjugate 36 wassuperior to an equivalent daily dose of hGH.

Abbreviations:

DBU 1,3-diazabicyclo[5.4.0]undecene

DCM dichloromethane

DIEA diisopropylethylamine

DMAP dimethylamino-pyridine

DMF N,N-dimethylformamide

DMSO dimethylsulfoxide

eq stoichiometric equivalent

fmoc 9-fluorenylmethoxycarbonyl

HFIP hexafluoroisopropanol

HOSu N-hydroxysuccinimide

LCMS mass spectrometry-coupled liquid chromatography

Mal maleimidopropionyl

MS mass spectrum

MW molecular mass

PEG polyethylene glycol

RP-HPLC reversed-phase high performance liquid chromatography

R_(f) retention factor

r.t. room temperature

SEC size exclusion chromatography

Suc succinimidopropionyl

TES triethylsilane

TFA trifluoroacetic acid

THF tetrahydrofurane

Trt trityl

REFERENCE LIST

1. Büyükgebiz A. et al J. Pediatr. Endocrinol. Metab. 1999January-February; 12(1):95-7

2. Clark et al, 1996, Journal of Biological Chemistry 271: 21969-21977

3. Girard, J. Mehls, O., J. Clin Invest. 1994 March; 93(3): 1163-1171

4. Philip Harris et al. Horn Res. 2006; 65 (suppl. 4): 1-213, CF1-98GH/IGF Treatment with title “First in-human study of PEGylatedrecombinant human growth hormone”.

5. Veronese, F. M. “Enzymes for Human Therapy: Surface StructureModifications,” Chimica Oggi, 7:53-56 (1989).

1. A prodrug conjugate of human growth hormone (hGH) of formula (I) or(II):

wherein: T represents hGH-NH; X represents a spacer moiety; Y₁ and Y₂each independently represent O, S, or NR₆; Y₃ and Y₅ represents O or S,independently of each other; Y₄ represents O, NR₆, or —C(R₇)(R₈); R₂ andR₃ represent independently of each other a moiety selected from thegroup consisting of hydrogen, substituted or unsubstituted linear,branched or cyclical alkyl or heteroalkyl groups, aryls, substitutedaryls, substituted or unsubstituted heteroaryls, cyano groups, nitrogroups, halogens, carboxy groups, carboxyalkyl groups, alkylcarbonylgroups, and carboxamidoalkyl groups; R₄ represents a moiety selectedfrom the group consisting of hydrogen, substituted or unsubstitutedlinear, branched or cyclical alkyls or heteroalkyls, aryls, substitutedaryls, substituted or unsubstituted heteroaryl, substituted orunsubstituted linear, branched or cyclical alkoxys, substituted orunsubstituted linear, branched or cyclical heteroalkyloxys, aryloxys orheteroaryloxys, cyano groups, and halogens; R₇ and R₈ are eachindependently selected from the group consisting of hydrogen,substituted or unsubstituted linear, branched or cyclical alkyls orheteroalkyls, aryls, substituted aryls, substituted or unsubstitutedheteroaryls, carboxyalkyl groups, alkylcarbonyl groups, carboxamidoalkylgroups, cyano groups, and halogens; R₆ represents a group selected fromthe group consisting of hydrogen, substituted or unsubstituted linear,branched or cyclical alkyls or heteroalkyls, aryls, substituted aryls,and substituted or unsubstituted heteroaryls; R₁ is a polymeric carrierhaving at least 3 chains S⁰, S¹, and S²; W represents a group selectedfrom the group consisting of substituted or unsubstituted linear,branched or cyclical alkyls, aryls, substituted aryls, substituted orunsubstituted linear, branched or cyclical heteroalkyls, and substitutedor unsubstituted heteroaryls; Nu represents a nucleophile; n representszero or a positive imager; and Ar represents a multi-substitutedaromatic hydrocarbon or multi-substituted aromatic heterocycle; andwherein the molecular weight of the prodrug without the hGH-NH is atleast 25 kDa and at most 1000 kDa.
 2. The prodrug of claim 1; whereinthe molecular weight of the prodrug without the hGH-NH is at least 25kDa and at most 500 kDa.
 3. The prodrug of claim 1; wherein themolecular weight of the prordrug without the hGH-NH is at least 30 andat most 250 kDa.
 4. The prodrug of claim 1; wherein the molecular weighof the prodrug without the hGH-NH is at least 30 kDa and at most 120kDa.
 5. The prodrug of claim 1; wherein the at least three chains S⁰,S¹, and S² are independently based on a polymer selected from the groupconsisting of polyalkyloxy polymers, hyaluronic acid and derivativesthereof, polyvinyl alcohols, polyoxazolines, polyanhydrides, poly(orthoesters), polycarbonates, polyurethanes, polyacrylic acids,polyacrylamides, polyacrylates, polymethacrylates,polyorganophosphazenes, polysiloxanes, polyvinylpyrrolidone,polycyanoacrylates, and polyesters.
 6. The prodrug of claim 1; whereinS⁰, S¹, and S² are based on a polyalkoxy polymer.
 7. The prodrug ofclaim 1; wherein S⁰, S¹, and S² are polyethylene glycol based.
 8. Theprodrug of claim 1; wherein the moiety

of formula (I) or (II) is selected from the group consisting of


9. The prodrug of claim 1; wherein the moiety

is selected from the group consisting of:


10. A clinical effective amount of the pharmaceutical composition ofclaim 1 for use in a method for treatment of growth hormone relateddisease in a human person.